Cell Surface Display, Screening and Production of Proteins of Interest

ABSTRACT

Aspects of the invention provide compositions and methods for displaying engineered polypeptides on a cell surface. According to aspects of the invention, immobilized polypeptides can be screened to identify one or more variants having one or more functional or structural properties of interest. Aspects of the invention provide composition and methods for producing engineered protein or protein variants having a functional or a structural property of interest.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/226,114, filed Mar. 26, 2014, now U.S. Pat. No. 9,645,146, issued onMay 9, 2017, which is a divisional of U.S. patent application Ser. No.12/056,264, filed Mar. 26, 2008, now U.S. Pat. No. 8,709,980, issued onApr. 29, 2014, which claims the benefit under 35 U.S.C. §119(e) fromU.S. provisional applications Ser. No. 60/920,378 entitled “In vivoprotein display as tools for surface display” filed Mar. 26, 2007, Ser.No. 60/920,375 entitled “Evaluating predetermined protein functions”,filed Mar. 26, 2007, Ser. No. 60/959,719 entitled “Cell surface displayof proteins”, filed Jul. 16, 2007, and Ser. No. 61/004,841 entitled“Cell surface display, screening and production of proteins of interest”filed Dec. 1, 2007, the entire contents of each which are hereinincorporated by reference in their entireties.

REFERENCE TO SEQUENCE LISTING

This specification includes a sequence listing, submitted herewith,which includes the file entitled “128255-011511DIV ST25.txt” having thefollowing size: 44,554 bytes which was created Mar. 26, 2014, thecontent of which is incorporated by reference herein.

FIELD OF THE INVENTION

The invention relates to the field of protein screening and librariesand components for protein screening. The invention also relates to thedisplay and production of proteins of interest.

BACKGROUND OF THE INVENTION

Recombinant and synthetic nucleic acids have many applications inresearch, industry, agriculture, and medicine. Recombinant and syntheticnucleic acids can be used to express and obtain large amounts ofpolypeptides, including enzymes, antibodies, growth factors, receptors,and other polypeptides that may be used for a variety of medical,industrial, or agricultural purposes. Methods for screening ofpolypeptides with a predetermined function or property have beendescribed, including phage display. However, current screening methodsare limited by the size of the libraries, the lengths and complexitiesof polypeptides and available functional assays.

SUMMARY OF THE INVENTION

Aspects of the invention relate to compositions and methods fordisplaying an engineered protein on a host cell surface. In someembodiments, an engineered protein is expressed in a host cell underconditions that result in the protein being modified in such a way thatthe modified protein binds to a surface-immobilized binding partner ifit is secreted from the host cell.

Accordingly, aspects of the invention are useful to express engineeredproteins from host cell nucleic acids so that engineered proteins aresecreted and retained on the surface of the host cells. Therefore,embodiments of the invention are useful to express and display manydifferent variant proteins and polypeptides on host cell surfaces and togenerate protein display libraries. These protein display libraries canbe used to screen for one or more structural and/or functionalproperties of the protein. Once a host cell is identified as having asurface-displayed protein with a desired characteristic or function, thenucleic acid that encodes that protein can be isolated andcharacterized.

Aspects of the invention include methods of expressing and displayingengineered proteins on host cells. Aspects of the invention includenucleic acid constructs, expressed proteins, host cells, and/or isolatedproteins.

In some embodiments, the invention provides a method for displaying anengineered protein on a host cell, the method comprising incubating ahost cell comprising a first nucleic acid under conditions sufficientfor expressing an engineered protein encoded by the first nucleic acid,wherein the host cell displays a first binding partner on its surface,wherein the engineered protein comprises a modification motif and asecond binding partner is coupled to the modification motif when theengineered protein is expressed and, wherein the expressed engineeredprotein is secreted from the host cell and displayed on the cell surfacevia binding of the second binding partner to the first binding partner.In some embodiments, the method further comprises displaying a pluralityof different engineered proteins, wherein each different engineeredprotein is encoded on a different first nucleic acid in a different hostcell. In some embodiments, the different engineered proteins aresequence variants of each other. In some embodiments, the engineeredprotein comprises a secretion peptide. In some embodiments, the hostcell is a yeast cell. In some embodiments, the modification motif is abiotin acceptor peptide. In some embodiments, the first binding partneris displayed via interaction with a further second binding partnerattached to the cell surface. In some embodiments, the first bindingpartner is an avidin-like protein. In some embodiments, the secondbinding partner is biotin. In some embodiments, coupling of the secondbinding partner to the modification motif is catalyzed by a couplingenzyme. In some embodiments, the coupling enzyme is encoded on a secondnucleic acid, wherein the second nucleic acid is a recombinant nucleicacid integrated into a vector or the genome of the host cell. In someembodiments, the coupling enzyme is a biotin ligase. In someembodiments, the method further comprises expressing a chaperone proteinin the host cell.

In some embodiments, the invention provides a method for displaying anengineered protein on a cell surface, the method comprising incubating ahost cell comprising a first nucleic acid under conditions sufficientfor expressing an engineered protein encoded by the first nucleic acid,wherein the host cell comprises a first binding partner on its surface,and wherein the first binding partner is attached to the cell surface bybinding to a cell wall protein comprising a second binding partner thatspecifically binds to the first binding partner, contacting the hostcell with a target molecule that also comprises the second bindingpartner under conditions sufficient to immobilize the target molecule onthe surface of the host cell via binding of the second binding partnerto the first binding partner, incubating the cells under conditionsresulting in secretion of the engineered protein, wherein the engineeredprotein binds to the target molecule, thereby displaying the engineeredprotein on the host cell surface. In some embodiments, the engineeredprotein is an antibody, a single chain antibody, a scaffold protein, ora fragment thereof.

In some embodiments, the invention provides a protein screening methodcomprising expressing an engineered protein comprising a modificationmotif in a host cell having a cell surface comprising a first bindingpartner, wherein a second binding partner is coupled to the expressedengineered protein, and wherein the expressed engineered protein issecreted and displayed on the cell surface via binding of the secondbinding partner to the first binding partner; and, evaluating a propertyof the engineered protein displayed on the cell surface. In someembodiments, the evaluating step comprises assaying a level of activity,determining whether the engineered protein has a predetermined function,comparing the property of the engineered protein to the property of areference protein, or determining the amount of the engineered proteindisplayed on the cell surface. In some embodiments, the engineeredprotein is an antibody, and the function of the antibody being evaluatedis the binding affinity of the antibody to the target molecule, whereinthe target molecule comprises an antigen and/or epitope. In someembodiments, the host cells are selected on the basis of a firstpredetermined property of the displayed engineered protein. In someembodiments, the method further comprises selecting the host cells onthe basis of a second predetermined property of the displayed engineeredprotein. In some embodiments, the method further comprises releasing theengineered protein from selected host cells displaying at least thepredetermined level of the engineered protein. In some embodiments,assaying a level of activity comprises assaying if the engineeredprotein can process a substrate. In some embodiments, the substrate iscoupled to the surface. In some embodiments, the substrate is apolypeptide, nucleic acid, lipid, polysaccharide, synthetic polymer orsynthetic compound. In some embodiments, processing a substratecomprises binding to the substrate, dissociating the substrate, nickingthe substrate, cutting the substrate, activating the substrate,deactivating the substrate, charging the substrate, decharging thesubstrate, changing substrate conformation, copying the substrate,replicating the substrate, conjugating molecules to the substrate,conjugating peptides to the substrate or modifying the substrate.

In some embodiments, the invention provides a method for evaluating ifan engineered protein can process a substrate, the method comprisinginducing expression of an engineered protein in a host cell, andmeasuring the level of a detectable signal generated by the engineeredprotein processing a substrate, wherein, a) the engineered protein issecreted, and b) the substrate is coupled to the host cell surface. Insome embodiments, the substrate is a polypeptide, nucleic acid, lipid,polysaccharide, synthetic polymer or synthetic compound. In someembodiments, processing a substrate comprises binding to the substrate,dissociating the substrate, nicking the substrate, cutting thesubstrate, activating the substrate, deactivating the substrate,charging the substrate, decharging the substrate, changing substrateconformation, copying the substrate, replicating the substrate,conjugating molecules to the substrate, conjugating peptides to thesubstrate or modifying the substrate.

In some embodiments, the invention provides a host cell that comprises afirst nucleic acid that encodes an engineered protein, wherein the hostcell is capable of having a first binding partner coupled to its cellsurface, wherein the engineered protein comprises a modification motif,and wherein expression of the engineered protein results in coupling ofa second binding partner to the modification motif and secretion of theengineered protein so that it can be displayed on the cell surface viainteraction binding of the second binding partner to the first bindingpartner. In some embodiments, the host cell displays at least 10²engineered proteins. In some embodiments, the host cell displays atleast 10³ engineered proteins. In some embodiments, the host celldisplays at least 10⁴ engineered proteins. In some embodiments, the hostcell displays at least 10⁵ engineered proteins, at least 10⁶ engineeredproteins or more. In some embodiments, the invention provides a libraryof any of the host cells described herein. In some embodiments, thelibrary has at least 10⁸ different members. In some embodiments, thelibrary has at least 2, at least 5, at least 10, at least 50, at least100, at least 1000, at least 10,000, at least 100,000, at least1,000,000, at least 10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰ or atleast 10¹¹ members.

In some embodiments, the invention provides a library of nucleic acids,the library comprising a plurality of nucleic acids, wherein eachnucleic acid encodes a different variant of an engineered protein, andwherein each variant comprises an identical modification motif capableof coupling a binding partner. In some embodiments, the modificationmotif is a biotinylation motif. In some embodiments, the library has atleast 10⁸ different members. In some embodiments, the library has atleast 2, at least 5, at least 10, at least 50, at least 100, at least1000, at least 10,000, at least 100,000, at least 1,000,000, at least10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰ or at least 10¹¹ members.

In some embodiments, the invention provides methods of isolating anengineered protein with a predetermined function or level of activity.

In some embodiments, the invention provides a method for displaying anengineered protein on a host cell, the method comprising incubating ahost cell comprising a first nucleic acid under conditions sufficientfor expressing an engineered protein encoded by the first nucleic acid,wherein the host cell displays a first binding partner, wherein theengineered protein comprises a modification motif and a second bindingpartner is coupled to the modification motif when the engineered proteinis expressed, and, wherein the expressed engineered protein is secretedfrom the host cell and displayed on the cell surface via binding of thesecond binding partner to the first binding partner. In someembodiments, the first binding partner is an avidin-like protein. Insome embodiments, the second binding partner is biotin. In someembodiments the modification motif is a biotinylation peptide. In someembodiments, coupling of the second binding partner is done by acoupling enzyme. In some embodiments, the coupling enzyme is a biotinligase. In some embodiments, the method further comprises expressing achaperone protein.

In some embodiments, the invention provides a method for displaying anengineered protein on a cell surface, the method comprising introducinga vector comprising a gene encoding an engineered protein in a hostcell, wherein the host cell displays a first binding partner on the cellsurface, coupling a target molecule attached to a second binding partnerto the first binding partner on the cell surface, thereby immobilizingthe target molecule on the cell surface, incubating the cells underconditions resulting in secretion of the engineered protein, wherein theengineered protein binds to the target molecule, thereby displaying theengineered protein on the host cell surface. In some embodiments, thefirst binding partner is attached to the cell surface through a cellwall protein. In some embodiments, the engineered protein is an antibodyand the target molecule is an antigen. In some embodiments, theengineered protein is a scaffold protein.

In some embodiments, the invention provides a method for displaying anengineered protein on a cell surface, the method comprising introducinga vector comprising a gene encoding an engineered protein in a hostcell, wherein the host cell displays a third binding partner on the cellsurface, adding a second binding partner to the host cell displaying thethird binding partner, wherein the second binding partner binds to thethird binding partner resulting in the display of the second bindingpartner on the cell surface, adding a target molecule attached to afirst binding partner to the second binding partner, wherein the secondbinding partner binds to the first binding partner resulting in thedisplay of the target molecule on the host cell surface, incubating thecells under conditions resulting in secretion of the engineered protein,wherein the engineered protein binds to the target molecule, therebydisplaying the engineered protein on the host cell surface.

In some embodiments, the invention provides a protein screening method,the method comprising expressing an engineered protein comprising amodification motif in a host cell having a cell surface comprising afirst binding partner, wherein a second binding partner is coupled tothe expressed engineered protein, and wherein the expressed engineeredprotein is secreted and displayed on the cell surface via binding of thesecond binding partner to the first binding partner; and, evaluating afunction of the engineered protein displayed on the cell surface. Insome embodiments, the evaluating step comprises assaying a level ofactivity, determining whether the engineered protein has a predeterminedfunction, or comparing the engineered protein to a reference protein. Insome embodiments, assaying a level of activity comprises assaying if theengineered protein can process a substrate. In some embodiments, thesubstrate is a polypeptide, nucleic acid, lipid, polysaccharide,synthetic polymer or synthetic compound. In some embodiments, process asubstrate comprises binding to the substrate, dissociating thesubstrate, nicking the substrate, cutting the substrate, activating thesubstrate, deactivating the substrate, charging the substrate,decharging the substrate, changing substrate conformation, copying thesubstrate, replicating the substrate, conjugating molecules to thesubstrate, conjugating peptides to the substrate or modifying thesubstrate.

In some embodiments, the invention provides a method for evaluating ifan engineered protein can process a substrate, the method comprisinginducing expression of an engineered protein in a host cell, andmeasuring the level of a detectable signal generated by the engineeredprotein processing a substrate, wherein a) the engineered protein issecreted, and b) the substrate is coupled to the host cell surface. Insome embodiments, the substrate is a polypeptide, nucleic acid, lipid,polysaccharide, synthetic polymer or synthetic compound. In someembodiments, processing a substrate comprises binding to the substrate,dissociating the substrate, nicking the substrate, cutting thesubstrate, activating the substrate, deactivating the substrate,charging the substrate, decharging the substrate, changing substrateconformation, copying the substrate, replicating the substrate,conjugating molecules to the substrate, conjugating peptides to thesubstrate or modifying the substrate.

In some embodiments, the invention provides a host cell that comprises afirst nucleic acid that encodes an engineered protein, wherein the hostcell is capable of having a first binding partner coupled to its cellsurface, wherein the engineered protein comprises a modification motif,and wherein expression of the engineered protein results in coupling ofa second binding partner to the modification motif and secretion of theengineered protein so that it can be displayed on the cell surface viainteraction binding of the second binding partner to the first bindingpartner. In some embodiments, the host cell displays at least 10⁴engineered proteins. In some embodiments, the invention provides libraryof host cells. In some embodiments, the library has at least 2, at least5, at least 10, at least 50, at least 100, at least 1000, at least10,000, at least 100,000, at least 1,000,000, at least 10⁷, at least10⁸, at least 10⁹, at least 10¹⁰ or at least 10¹¹ members.

In some embodiments, the invention provides a library of nucleic acids,the library comprising a plurality of nucleic acids, wherein eachnucleic acid encodes a different variant of an engineered protein, andwherein each variant comprises an identical modification motif. In someembodiments, the library has at least 2, at least 5, at least 10, atleast 50, at least 100, at least 1000, at least 10,000, at least100,000, at least 1,000,000, at least 10⁷, at least 10⁸, at least 10⁹,at least 10¹⁰ or at least 10¹¹ members.

In some embodiments, the invention provides a method for displaying anengineered protein on a cell surface, the method comprising introducinga vector comprising a gene encoding an engineered protein in a hostcell, wherein the host cell displays a first binding partner on the cellsurface, wherein the engineered protein comprises a modification motifexpressing the engineered proteins under conditions sufficient forcoupling a second binding partner to the modification motif, secretingthe engineered protein to the host cell surface; and, binding the secondbinding partner to the first binding partner on the cell surface,thereby displaying the engineered protein on the host cell surface.

In some embodiments, the invention provides a method for displaying anengineered protein on a cell surface, the method comprising generating ahost cell displaying a first binding partner at its cell surface,introducing a nucleic acid encoding an engineered protein comprising asequence encoding for a second binding partner or a modification motifallowing for in vivo coupling of a second binding partner, and,incubating the host cell under conditions sufficient for secretingengineered protein, wherein the first binding partner binds the secondbinding partner, thereby displaying the engineered protein on the hostcell surface.

In some embodiments, the invention provides a method for displaying anengineered protein on a cell surface, the method comprising generating ahost cell displaying a first binding partner on the cell surface,introducing a vector comprising a gene encoding an engineered proteincomprising a second binding partner, incubating the host cell underconditions sufficient for secreting the engineered protein, and bindingthe second binding partner to the first binding partner on the cellsurface, thereby displaying the engineered protein on the host cellsurface.

In some embodiments, the invention provides a method for displaying anengineered protein on a cell surface, the method comprising introducinga vector comprising a gene encoding an engineered protein in a hostcell, wherein the host cell displays a first binding partner on the cellsurface, coupling the target molecule to the first binding partner onthe cell surface, thereby immobilizing the target molecule on the cellsurface, inducing secretion of the engineered protein, and binding theengineered protein to the target molecule, thereby displaying theengineered protein on the host cell surface, wherein the first bindingpartner is connected to the cell surface through binding to a secondbinding partner that is attached to the cell surface.

In some embodiments, the invention provides a method for generating alibrary of engineered proteins displayed on a cell surface, the methodcomprising introducing a plurality of vectors into a population of hostcells, wherein the host cell displays a first binding partner on thecell surface, wherein each vector comprises a gene encoding a uniqueengineered protein, wherein each engineered protein comprises a uniquepolypeptide linked to an immobilization peptide, wherein theimmobilization peptide comprises a modification motif, expressing theengineered proteins under conditions sufficient for: a) coupling asecond binding partner to the modification motif; b) secreting theengineered proteins to the host cell surfaces, and, binding the secondbinding partner to the first binding partner on the cell surface,thereby generation of a library of engineered proteins displayed on acell surface.

In some embodiments, the invention provides a method for generating alibrary of engineered proteins displayed on a cell surface, the methodcomprising introducing a plurality of vectors into a population of hostcells, wherein the host cell displays a first binding partner on thecell surface, wherein each vector comprises a gene encoding a uniqueengineered protein linked to a second binding partner, expressing theengineered proteins under conditions sufficient for secreting theengineered proteins, and binding the second binding partner to the firstbinding partner on the cell surface, thereby generation of a library ofengineered proteins displayed on a cell surface.

In some embodiments, the invention provides a method for generating alibrary of engineered proteins on a cell surface, the method comprisingintroducing a plurality of vectors into a population of host cells,wherein the host cell displays a first binding partner on the cellsurface, wherein each vector comprises a gene encoding a uniqueengineered protein, wherein the engineered protein has affinity to atarget molecule, coupling the target molecule to the first bindingpartner on the cell surface, thereby immobilizing the target molecule onthe cell surface, inducing secretion of the engineered proteins, and,binding the engineered protein to the target molecule, therebygeneration of a library of engineered proteins displayed on a cellsurface.

In some embodiments, the invention provides a method for evaluating ifan engineered protein has a predetermined function, the methodcomprising inducing expression of an engineered protein in a host cell,wherein the host cell displays a first binding partner on the cellsurface, and wherein a) the engineered protein comprises a modificationmotif; b) a second binding partner is coupled to the modification motif;c) the engineered protein is secreted; and, d) the second bindingpartner is bound to the first binding partner on the cell surface,thereby displaying the engineered protein on the host cell surface, andevaluating if the engineered protein has the predetermined function.

In some embodiments, the invention provides a method for assaying theactivity of an engineered protein, the method comprising inducingexpression of an engineered protein in a host cell, wherein the hostcell displays a first binding partner on the cell surface, and whereina) the engineered protein comprises a modification motif; b) a secondbinding partner is coupled to the modification motif; c) the engineeredprotein is secreted; and, d) the second binding partner is bound to thefirst binding partner on the cell surface, thereby displaying theengineered protein on the host cell surface, and assaying the activityof the engineered protein.

In some embodiments, the invention provides a method for evaluating if aprotein complex of engineered proteins has a predetermined function, themethod comprising inducing expression of two or more engineered proteinsin a host cell, wherein the host cell displays a first binding partneron the cell surface, and wherein a) the engineered protein comprises amodification motif; b) a second binding partner is coupled to themodification motif; c) the engineered protein is secreted; and d) thesecond binding partner is bound to the first binding partner on the cellsurface, thereby displaying the engineered protein on the host cellsurface, wherein the two or more engineered proteins interact to form aprotein complex of engineered proteins, and evaluating if the proteincomplex of engineered proteins has the predetermined function.

In some embodiments, the invention provides a method for assaying theactivity of a protein complex of engineered proteins, the methodcomprising inducing expression of two or more engineered proteins in ahost cell, wherein the host cell displays a first binding partner on thecell surface, and wherein a) the engineered protein comprises amodification motif; b) a second binding partner is coupled to themodification motif; c) the engineered protein is secreted; and, d) thesecond binding partner is bound to the first binding partner on the cellsurface, thereby displaying the engineered protein on the host cellsurface, wherein the two or more engineered proteins interact to form aprotein complex of engineered proteins, and assaying the activity of theprotein complex of engineered proteins.

In some embodiments, the invention provides a method for evaluating ifan engineered protein can process a substrate, the method comprisinginducing expression of an engineered protein in a host cell, wherein thehost cell displays a first binding partner on the cell surface, andwherein a) the engineered protein comprises a modification motif; b) asecond binding partner is coupled to the modification motif; c) theengineered protein is secreted; and, d) the second binding partner isbound to the first binding partner on the cell surface, therebydisplaying the engineered protein on the host cell surface; andcontacting the engineered protein with a substrate, wherein if theengineered protein interacts with the substrate, the engineered proteincan process the substrate.

In some embodiments, the invention provides a method for assaying theactivity of an engineered protein, the method comprising inducingexpression of an engineered protein in a host cell, wherein the hostcell displays a first binding partner on the cell surface, and whereina) the engineered protein comprises a modification motif; b) a secondbinding partner is coupled to the modification motif; c) the engineeredprotein is secreted; and, d) the second binding partner is bound to thefirst binding partner on the cell surface, thereby displaying theengineered protein on the host cell surface; contacting the engineeredprotein with a substrate, and assaying the activity of the engineeredprotein.

In some embodiments, the invention provides a method for evaluating if aprotein complex of engineered proteins can process a substrate, themethod comprising inducing expression of two or more engineered proteinsin a host cell, wherein the host cell displays a first binding partneron the cell surface, and wherein a) the engineered protein comprises amodification motif; b) a second binding partner is coupled to themodification motif; c) the engineered protein is secreted; and, d) thesecond binding partner is bound to the first binding partner on the cellsurface, thereby displaying the engineered protein on the host cellsurface, wherein the two or more engineered proteins interact to form aprotein complex of engineered proteins, and contacting the proteincomplex of engineered proteins with a substrate, wherein if the proteincomplex of engineered proteins interacts with the substrate, the proteincomplex of engineered proteins can process the substrate.

In some embodiments, the invention provides a method for assaying theactivity of a protein complex of engineered proteins, the methodcomprising inducing expression of two or more engineered proteins in ahost cell, wherein the host cell displays a first binding partner on thecell surface, and wherein a) the engineered protein comprises amodification motif; b) a second binding partner is coupled to themodification motif; c) the engineered protein is secreted; and, d) thesecond binding partner is bound to the first binding partner on the cellsurface, thereby displaying the engineered protein on the host cellsurface, wherein the two or more engineered proteins interact to form aprotein complex of engineered proteins, contacting the protein complexof engineered proteins with a substrate; and, assaying the activity ofthe protein complex of engineered proteins.

In some embodiments, the invention provides a method for evaluating ifan engineered protein can process a substrate, the method comprisinginducing expression of an engineered protein in a host cell, andmeasuring the level of a detectable signal generated by the engineeredprotein processing a substrate, wherein, a) the engineered protein issecreted, and b) the substrate is coupled to the host cell surface.

In some embodiments, the invention provides a method for screeningcandidate engineered proteins, the method comprising introducing aplurality of vectors into a population of host cells, wherein the hostcell displays a first binding partner on the cell surface, wherein eachvector comprises a gene encoding a unique engineered protein, inducingexpression of the engineered proteins in host cells, and wherein a) theengineered protein comprises a modification motif; b) a second bindingpartner is coupled to the modification motif; c) the engineered proteinis secreted; and d) the second binding partner is bound to the firstbinding partner on the cell surface, thereby displaying the engineeredprotein on the host cell surface, and determining if the engineeredproteins have a predetermined function, wherein if the engineeredprotein has the predetermined function, the engineered protein isidentified as a candidate engineered protein.

In some embodiments, the invention provides a method for screeningcandidate engineered proteins, the method comprising introducing aplurality of vectors into a population of host cells, wherein the hostcell displays a first binding partner on the cell surface, wherein eachvector comprises a gene encoding a unique engineered protein, inducingexpression of the engineered proteins in host cells, and wherein a) theengineered protein comprises a modification motif; b) a second bindingpartner is coupled to the modification motif; c) the engineered proteinis secreted; and, d) the second binding partner is bound to the firstbinding partner on the cell surface, thereby displaying the engineeredprotein on the host cell surface, and contacting the engineered proteinswith a substrate, wherein if an engineered protein interacts with thesubstrate, the engineered protein is identified as a candidateengineered protein.

In some embodiments, the invention provides a method for screeningcandidate engineered proteins, the method comprising introducing aplurality of vectors into a population of host cells, wherein eachvector comprises a gene encoding a unique engineered protein, inducingexpression of the engineered proteins in the host cells, wherein a) theengineered proteins are secreted, and b) a substrate is coupled to thehost cell surfaces, wherein if the engineered protein processes thesubstrate a detectable signal is generated, and wherein if a detectablesignal is generated the engineered protein is identified as a candidateengineered protein.

In some embodiments, the invention provides a method for producing anengineered protein having a predetermined affinity for a targetmolecule, the method comprising introducing a plurality of vectors intoa population of host cells, wherein the host cell displays a firstbinding partner on the cell surface, wherein each vector comprises agene encoding a unique engineered protein, coupling a target molecule tothe first binding partner, thereby immobilizing the target molecule onthe cell surface, inducing secretion of the engineered proteins, bindingthe engineered proteins to the target molecule on the host cell surfaceresulting in the generation of a library of engineered proteins on thehost cell surfaces, selecting a subpopulation of host cells secreting anengineered protein of interest having a predetermined affinity for thetarget molecule, and producing the engineered protein from the selectedsubpopulation of host cells.

In some embodiments, the invention provides a library of host cells,wherein each host cell comprises a vector encoding a gene for a uniqueengineered protein and wherein the host cell also comprises a substrate,and wherein the substrate is coupled to the host cell surface. In someembodiments, the invention provides a library of host cells, whereineach host cell expresses a unique engineered protein and comprises asubstrate, wherein the substrate is coupled to the host cell surface. Insome embodiments, the invention provides a library of host cells,wherein each host cell expresses a unique engineered protein and whereineach engineered protein comprises a unique polypeptide coupled to animmobilization peptide. In some embodiments, the invention provides alibrary of host cells, wherein each host cell displays on its surface anengineered protein and wherein each engineered protein comprises aunique polypeptide coupled to an immobilization peptide. In someembodiments, the invention provides a library of vectors, wherein eachvector comprises a nucleic acid encoding a unique engineered protein andwherein each engineered protein comprises a unique polypeptide coupledto an immobilization peptide. In some embodiments, the inventionprovides a library of engineered proteins, wherein each engineeredprotein comprises a unique polypeptide coupled to an immobilizationpeptide.

It should be appreciated that in any of the embodiments theimmobilization peptide can be linked to the C-terminus or the N-terminusof the engineered protein. It should be appreciated that in any of theembodiments the immobilization peptide can be linked to the N-terminusof the engineered protein. It should be appreciated that in any of theembodiments the engineered protein can comprise a secretion peptide. Itshould be appreciated that in any of the embodiments the gene encodingthe engineered protein may be integrated in the genome of the cell.

It should be appreciated that in any of the embodiments the engineeredprotein can comprise a therapeutic polypeptide, polymerase, ligase,restriction enzyme, topoisomerase, kinase, phosphatase, metabolicenzyme, catalytic enzyme, therapeutic enzyme, pharmaceutical enzyme,environmental enzyme, industrial enzyme, pharmaceutical polypeptide,environmental polypeptide, industrial polypeptide, binding protein,antibody, antibody fragment, signaling molecule, cytokine or a receptor.It should be appreciated that in any of the embodiments the engineeredprotein can comprise a polymerase. It should be appreciated that in anyof the embodiments the polymerase can be a phi 29 polymerase.

It should be appreciated that in any of the embodiments the engineeredprotein can be a scaffold protein. It should be appreciated that in anyof the embodiments the engineered protein can comprise a reportermoiety.

It should be appreciated that in any of the embodiments theimmobilization peptide can be a transmembrane polypeptide. It should beappreciated that in any of the embodiments the immobilization peptidecan be a polypeptide membrane anchor. It should be appreciated that inany of the embodiments the immobilization peptide can be a GPI-linkedpolypeptide. It should be appreciated that in any of the embodiments theimmobilization peptide can be a natural surface polypeptide. It shouldbe appreciated that in any of the embodiments the natural surfacepolypeptide can be Aga2.

It should be appreciated that in any of the embodiments the coupling thesecond binding partner can be done in vivo by a coupling enzyme. Itshould be appreciated that in any of the embodiments the coupling thesecond binding partner can be catalyzed in vivo by a coupling enzyme. Itshould be appreciated that in any of the embodiments the method canfurther comprise expressing a coupling enzyme. It should be appreciatedthat in any of the embodiments the coupling enzyme can be expressed froma vector. It should be appreciated that in any of the embodiments thegene encoding the coupling enzyme can be integrated in the genome of thecell. It should be appreciated that in any of the embodiments thecoupling enzyme can be biotin ligase. It should be appreciated that inany of the embodiments the coupling enzyme can be BirA. It should beappreciated that in any of the embodiments the coupling enzyme can beBpl1.

It should be appreciated that in any of the embodiments the modificationmotif can be a biotinylation peptide. It should be appreciated that inany of the embodiments the biotinylation peptide can have a sequencerecognized by BirA. It should be appreciated that in any of theembodiments the second binding partner can be biotin.

It should be appreciated that in any of the embodiments the secondbinding partner can be a carbohydrate binding domain. It should beappreciated that in any of the embodiments the carbohydrate bindingdomain can be a lectin. It should be appreciated that in any of theembodiments the lectin can be concanavalin A. It should be appreciatedthat in any of the embodiments the lectin can be Phytohemaglutinin. Itshould be appreciated that in any of the embodiments the carbohydratebinding domain can be the sugar-binding domain of a flocculationprotein. It should be appreciated that in any of the embodiments theflocculation protein can be selected from the group consisting of Flo1,Flo5 and Flo11. It should be appreciated that in any of the embodimentsthe carbohydrate binding domain can be a carbohydrate binding module. Itshould be appreciated that in any of the embodiments the carbohydratebinding module can be a cellulose binding domain.

It should be appreciated that in any of the embodiments the method canfurther comprise introducing a vector comprising a gene encoding thefirst binding partner in the host cells, and incubating the host cellsunder conditions sufficient for expressing the first binding partner onthe host cell surface. It should be appreciated that in any of theembodiments the first binding partner can be avidin, streptavidin orneutravidin. It should be appreciated that in any of the embodiments thefirst binding partner can comprise at least one monomer of an avidin oravidin like protein. It should be appreciated that in any of theembodiments the first binding partner can be a fusion protein. It shouldbe appreciated that in any of the embodiments the fusion protein cancomprise an anchoring motif. It should be appreciated that in any of theembodiments the anchoring motif can be selected from the groupconsisting of GPI anchor, modified GPI anchor, α-agglutinin,a-agglutinin, flocculation protein, major cell wall proteins, CCW14,CIS3, CWP1, PIR1, and PIR3. It should be appreciated that in any of theembodiments the fusion protein can comprise a secretion peptide. Itshould be appreciated that in any of the embodiments the first bindingpartner can be expressed as a single polypeptide. It should beappreciated that in any of the embodiments the first binding partner canbe connected to the cell surface through a biotin spacer. It should beappreciated that in any of the embodiments the biotin spacer cancomprise a PEG moiety. It should be appreciated that in any of theembodiments the first binding partner can be biotin. It should beappreciated that in any of the embodiments in the first binding partnercan be a biotin binding protein or portion thereof.

It should be appreciated that in any of the embodiments the conditionsfor expressing the engineered protein and the first binding partner canbe different. It should be appreciated that in any of the embodimentsthe conditions for expressing the engineered protein and the firstbinding partner can be the same. It should be appreciated that in any ofthe embodiments the condition can comprise the addition of an agent tothe environment of the host cell. It should be appreciated that in anyof the embodiments the condition can comprise a change in temperature ofthe environment of the host cell. It should be appreciated that in anyof the embodiments the condition can comprise a change in carbon sourceof the host cell. It should be appreciated that in any of theembodiments the first binding partner can be covalently conjugated tothe cell surface. It should be appreciated that in any of theembodiments the method can further comprise the expression of achaperone polypeptide.

It should be appreciated that in any of the embodiments the cell can bea mammalian cell. It should be appreciated that in any of theembodiments the cell can be a yeast cell. It should be appreciated thatin any of the embodiments the cell can be S. cerevisiae. It should beappreciated that in any of the embodiments the cell can be a bacterialcell. It should be appreciated that in any of the embodiments the cellcan be E. coli. It should be appreciated that in any of the embodimentsthe cell can be a cell that has a reduced level of protease activity. Itshould be appreciated that in any of the embodiments the cell can beexpressing a chaperone protein.

It should be appreciated that in any of the embodiments the substratecan be coupled to the cell surface. It should be appreciated that in anyof the embodiments the substrate can be a polypeptide, nucleic acid,lipid, polysaccharide, synthetic polymer or synthetic compound. Itshould be appreciated that in any of the embodiments the substrate canbe coupled to the cell prior to inducing expression. It should beappreciated that in any of the embodiments the substrate can be coupledto the cell after expression has been induced. It should be appreciatedthat in any of the embodiments the substrate can be a moiety notnaturally present on the host cell surface. It should be appreciatedthat in any of the embodiments the substrate can be coupled to a moietynaturally present on the host cell surface. It should be appreciatedthat in any of the embodiments the substrate can be coupled to an aminoacid side chain of a polypeptide. It should be appreciated that in anyof the embodiments the substrate can comprises biotin. It should beappreciated that in any of the embodiments the substrate can beimmobilized on the cell surface through binding of the biotin to abinding moiety. It should be appreciated that in any of the embodimentsthe substrate can comprise a nucleic acid. It should be appreciated thatin any of the embodiments the substrate can comprise a template and aprimer.

It should be appreciated that in any of the embodiments processing ofthe substrate can comprise binding to the substrate, dissociating thesubstrate, nicking the substrate, cutting the substrate, activating thesubstrate, deactivating the substrate, charging the substrate,decharging the substrate, changing substrate conformation, copying thesubstrate, replicating the substrate, conjugating molecules to thesubstrate, conjugating peptides to the substrate or modifying thesubstrate. It should be appreciated that in any of the embodiments thethreshold level can be the level of signal of a control polypeptide. Itshould be appreciated that in any of the embodiments the method canfurther comprise contacting the engineered proteins with a substrateresults in a signal, and wherein if the level of the signal is above athreshold level, the engineered protein is identified as a candidateengineered protein.

It should be appreciated that in any of the embodiments the controlpolypeptide or control protein can be a polypeptide with random coilstructure. It should be appreciated that in any of the embodiments thecontrol polypeptide or protein can be a wild type polypeptide. It shouldbe appreciated that in any of the embodiments the control polypeptide orcontrol protein can be a commercially available polypeptide. It shouldbe appreciated that in any of the embodiments substrate processing cancomprise adding one or more nucleotides to a primer. It should beappreciated that in any of the embodiments substrate processing cancomprise adding one or more nucleotide comprising a fluorescent moietyto a primer. It should be appreciated that in any of the embodiments themethod can further comprise comparing the detectable signal to a signalof a control protein. It should be appreciated that in any of theembodiments the act of isolating the candidate engineered polypeptidecan be based on identifying a detectable signal above a threshold level.It should be appreciated that in any of the embodiments the detectablesignal can be generated by the engineered protein.

It should be appreciated that in any of the embodiments the detectablesignal can be generated by processing the substrate. It should beappreciated that in any of the embodiments the detectable signal can begenerated both by the engineered protein and by processing thesubstrate. It should be appreciated that in any of the embodiments thedetectable signal can be a fluorescent signal. It should be appreciatedthat in any of the embodiments the act of isolating the candidateengineered polypeptide can comprise a fluorescence activated cellsorting act.

It should be appreciated that the invention also comprises the candidateengineered proteins identified by any of the embodiments of theinvention.

It should be appreciated that in any of the embodiments the engineeredprotein can be an antibody or an antibody fragment and the targetmolecule can be an antigen. It should be appreciated that in any of theembodiments the engineered protein can be an antigen and the targetmolecule can be an antibody or antibody fragment. It should beappreciated that in any of the embodiments the engineered protein can bean antigen. It should be appreciated that in any of the embodiments theengineered protein can be an antibody or antibody fragments. It shouldbe appreciated that in any of the embodiments the engineered protein canbe a receptor and the target molecule can be a ligand.

It should be appreciated that in any of the embodiments the library canhave at least 2, at least 5, at least 10, at least 50, at least 100, atleast 1000, at least 10,000, at least 100,000, at least 1,000,000, atleast 10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰ or at least 10¹¹members. It should be appreciated that in any of the embodiments thehost cell can displays at least 10⁴ engineered proteins per cell. Itshould be appreciated that in any of the embodiments the immobilizationpeptide can be a biotinylation peptide. It should be appreciated that inany of the embodiments the immobilization peptide can be a transmembranepolypeptide. It should be appreciated that in any of the embodiments theimmobilization peptide can be a polypeptide membrane anchor. It shouldbe appreciated that in any of the embodiments the immobilization peptidecan be a GPI polypeptide. It should be appreciated that in any of theembodiments the immobilization peptide can be a natural surfacepolypeptide.

It should be appreciated that in any of the embodiments the reportermoiety can be a fluorescent protein. It should be appreciated that inany of the embodiments the method can further comprise binding of adetectable agent to the reporter moiety. It should be appreciated thatin any of the embodiments the detectable agent can be an antibody. Itshould be appreciated that in any of the embodiments the antibody cancomprise a fluorescent moiety.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a non-limiting overview of the expression of anengineered protein comprising a protein of interest linked to abiotinylation peptide (a second binding partner) wherein avidin (thefirst binding partner) is linked to the cell surface by a linker.

FIG. 2 illustrates a non-limiting overview of the expression of anengineered protein comprising a protein of interest linked to abiotinylation peptide (a second binding partner) wherein avidin (thefirst binding partner) is linked to the cell surface directly.

FIGS. 3A-3C show an embodiment where avidin is conjugated covalently tocells. FIG. 3A shows a negative control of labeling with biotinylatedfluorescence. FIG. 3B shows the conjugation with avidin through labelingwith biotinylated fluorescence. FIG. 3C shows the stability of theavidin-cell connection over time.

FIGS. 4A-4D illustrate an embodiment where an engineered cell wallprotein fused to two biotin acceptor peptides (BAP) is expressed at thecell surface. FIG. 4A shows a biotinylated engineered cell wall proteinexpressed at the cell surface. FIG. 4B shows the avidin binding to thebiotinylated cell wall protein. FIG. 4C shows the binding ofavidin-fluorescein to populations of cells expressing or not expressingthe biotinylated engineered cell wall protein. FIG. 4D shows the bindingof biotin-fluorescein to the avidin bound to the biotinylated engineeredcell wall protein.

FIGS. 5A-5F illustrate an embodiment of the expression and display of aprotein of interest at a cell surface. FIG. 5A illustrates a celltransformed with a vector encoding an engineered protein comprising aprotein of interest fused to biotin acceptor protein (BAP) and to anepitope tag. FIG. 5B illustrates biotinylation of the cell surface. FIG.5C illustrates the binding of avidin to the biotin at the cell surface.FIG. 5D illustrates the expression of the protein of interest fused tothe BAP and the epitope-tag (FLAG) and the expression of the biotinligase. FIG. 5E illustrates the in vivo biotinylation of the engineeredprotein. FIG. 5F illustrates the secretion of the engineeredbiotinylated protein and its binding to the avidin displayed at the cellsurface.

FIGS. 6A-6C illustrate an embodiment of the display of a target moleculeon the cell surface. FIG. 6A illustrates the binding of an antigen to anantibody (a target molecule) displayed at the cell surface through itsbinding to avidin, which in turn is bound to the biotin at the cellsurface. FIG. 6B illustrates the labeling of the antigen. FIG. 6Cillustrates the labeling of both the antigen and the displayed antibody.

FIGS. 7A-7D show a non-limiting embodiment of the expression of an scFvprotein. FIG. 7A illustrates the constructs encoding an scFv proteincomprising a promoter, a secretory leader sequence (SL), an epitope-Tag,the ORF encoding the scFv protein, a biotin acceptor peptide (BAP) and amating factor alpha terminator sequence. FIG. 7B illustrates theconstructs encoding an scFv protein comprising a promoter, a secretoryleader sequence (SL), a biotin acceptor peptide (BAP), the ORF encodingthe scFv protein, an epitope-Tag, and a mating factor alpha terminatorsequence. FIG. 7C illustrates the scFv protein with a BAP at itsC-terminus and a FLAG epitope at its N-terminus. FIG. 7D illustrates thescFv protein with a FLAG epitope at its C-terminus and a BAP at itsN-terminus.

FIGS. 8A-8D show a non-limiting embodiment of the display of the scFvprotein at the cell surface and the selection of the cells expressingthe scFv protein bound to an antigen. FIG. 8A illustrates the expressionof the scFv protein and the labeling with an antibody recognizing theFLAG epitope and the HIS6 of the antigen. FIG. 8B shows an assay for theenrichment of a population of cells expressing the antigen-binding scFvprotein. FIG. 8C shows an assay for the enrichment of a population ofcells expressing the antigen-binding scFv protein. FIG. 8D shows theenrichment of a population of cells expressing the antigen-binding scFvprotein.

FIGS. 9A-9B show a non-limiting embodiment of a display of an antibody.FIG. 9A shows an embodiment where the antibody is expressed at the cellsurface. FIG. 9B shows an assay for the enrichment of a population ofcells expressing the antibody through antigen binding.

FIGS. 10A shows a non limiting embodiment of the selection ofthermostable protein mutants. FIG. 10B shows a non limiting embodimentof the fold enrichment of thermostable protein mutants.

FIG. 11A shows a non-limiting embodiment of antibody expression anddisplay at the cell surface. FIG. 11B shows a non-limiting embodiment ofantibody expression and display at the cell surface and enrichment ofthe antibody population. FIG. 11C shows a non-limiting embodiment ofantibody expression and display at the cell surface and enrichment ofthe antibody population.

FIGS. 12A-12D show a non-limiting embodiment of protein display and cellcycle inhibition. FIG. 12A shows binding of biotin-fluorescein to avidinbound to a biotinylated cell wall protein in the absence of nocodazole.FIG. 12B shows labeling with the antigen and anti-FLAG antibody in theabsence of nocodazole. FIG. 12C shows binding of biotin-fluorescein toavidin bound to a biotinylated cell wall protein in the presence ofnocodazole. FIG. 12D shows labeling with the antigen and anti-FLAGantibody in the presence of nocodazole.

FIG. 13 shows a non-limiting embodiment of a substrate displayed on acell. IN one embodiment a cell labeled with NHS-PEG-biotin orhydrazine-PEG-biotin is provided. Upon subsequent addition and bindingof avidin, a biotinylated oligonucleotide is added and immobilized onthe cell surface. In a next step a complementary strand or extensionprimer can be supplied. In on embodiment, enzymatic chemistry can beperformed on the surface linked DNA construct by surface expressedenzymes.

FIG. 14A shows a non-limiting embodiment of a fluorescent signal of anassay comprising a single-stranded oligonucleotide immobilized on thesurface of yeast with an avidin sandwich. A FAM labelednon-complementary oligonucleotide was added to the cells. Specificbinding of the complementary oligonucleotide is shown by FITCfluorescence. FIG. 14B shows a non-limiting embodiment of a fluorescentsignal of an assay comprising a single-stranded oligonucleotideimmobilized on the surface of yeast with an avidin sandwich. A FAMlabeled complementary oligonucleotide was added to the cells. Specificbinding of the complementary oligonucleotide is shown by FITCfluorescence.

FIG. 15 shows a non-limiting embodiment depicting the decrease of afluorescent signal over time of a surface attached FAM labeled doublestranded oligonucleotide comprising an I-SceI restriction site, whichwas incubated with NheI or SceI. The resulting oligonucleotide cleavagewas indicated by decreased FAM fluorescence; and,

FIG. 16A shows a non-limiting embodiment of a fluorescent signal of aprimer that was extended on a single-stranded DNA template attached tothe surface of yeast. The fluorescent signal shows the incorporation ofCy-5 labeled dNTP by Klenow fragment. FIG. 16B shows a non-limitingembodiment of a fluorescent signal of a primer in the absence of Klenowfragment.

DETAILED DESCRIPTION

Aspects of the invention relate to methods and compositions fordisplaying one or more cellularly expressed molecules on a cell surface.In some embodiments, the molecules (e.g., engineered proteins) areexpressed in host cells under conditions resulting in theirimmobilization (e.g., the immobilization of the engineered proteins) onthe cell surface. Aspects of the invention are useful to immobilize oneor more proteins on a cell in which they are synthesized, therebyassociating each of the one or more proteins with their genotype. Thedisplayed protein(s) may be interrogated using any suitable assay orexperimental system to evaluate one or more properties or functions ofthe protein (e.g., binding properties, enzymatic activity, stability,etc., or any combination thereof). Preferred assays maintain theintegrity of the host cell so that the genotype of a protein beingevaluated can be retrieved (e.g., by characterizing the nucleic acidthat encoded the protein) if desired. In some embodiments, a singleprotein of interest may be displayed and evaluated using compositionsand methods of the invention. In certain embodiments, a library ofdifferent proteins may be evaluated. Each different protein may beencoded by a different nucleic acid in a different host cell of thelibrary. The different proteins may be variants that are being evaluatedto identify or select for novel and/or improved properties (e.g.,increased activity, increased stability, increased binding affinity,etc., or any combination thereof). In some embodiments, the variantsshare a common amino acid sequence for much of the protein, but differin amino acid sequence in areas that are suspected of being structurallyand/or functionally important. For example, antibody variants may shareone or more common framework sequences, but have different variablesequences (e.g., with different CDR sequences). Receptor libraries mayinclude receptor variants with different amino acid sequences in theirligand binding domains. Similarly, enzyme libraries may include enzymevariants with different sequences in their active site regions. Aspectsof the invention may be used to display and evaluate large numbers ofvariants so that a large sequence space may be sampled and tested. Insome embodiments, each host cell expresses and displays only one type ofprotein variant to be evaluated. However, aspects of the invention maybe useful to express and display many copies of the protein on the cellsurface. The high capacity of display systems of the invention is usefulto effectively and efficiently assay the displayed protein (e.g., inhigh throughput assays). In certain embodiments, a single host cell mayexpress and display two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) different protein variants (e.g., for evaluating pools ofproteins).

Other aspects of the invention relate to methods and compositions forproducing a molecule of interest in a cell. In a preferred embodiment,the molecule of interest is a protein or a polypeptide. As used herein,the terms “protein” and “polypeptide” are used interchangeably. The term“engineered protein” encompasses naturally occurring proteins andsynthetic polypeptides and protein constructs that comprise a syntheticpolypeptide or naturally occurring protein linked to additionalpolypeptide elements, like, for instance, an immobilization peptide,reporter peptide or secretion peptide. Proteins may or may not be madeup entirely of amino acids. Examples of classes of proteins that includeother non amino acid constituents include, but are not limited to,glycoproteins, lipoproteins and proteoglycans. According to aspects ofthe invention, engineered proteins are encoded and/or expressed from arecombinant nucleic acid that may be engineered to include sequencevariants, recombinant promoters, transcriptional control elements,fusion peptides, other modifications, or any combination of two or morethereof. In some embodiments proteins are displayed on the cell surfaceby immobilization of the protein. In some embodiments of the invention,proteins may be immobilized on a cell surface via a peptide that ismembrane or surface bound. In certain embodiments, immobilization mayinvolve specific interactions between binding partners. For example, aprotein of interest is attached to a host cell surface through theinteraction between a first and a second binding partner. In someembodiments, a first binding partner may be attached at the host cellsurface and an engineered protein may include a second binding partnerthat interacts specifically with the first binding partner (e.g., with abinding affinity represented by a dissociation constant of about 10⁻⁷M,about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M, about 10⁻¹² M,about 10⁻¹³ M, about 10⁻¹⁴ M or about 10⁻¹⁵ M). In some embodiments,engineered proteins comprise a modification motif that is modified by acoupling enzyme, resulting in the coupling of a second binding partnerto the modification motif. In some embodiments, the second bindingpartner is coupled to the engineered protein intracellularly.

In some embodiments, a target molecule (e.g., a target protein) isattached to the cell surface directly and the expressed engineeredprotein binds to the target protein, thereby displaying the expressedprotein. In some embodiments, the target molecule is not attacheddirectly to the host cell but is attached to a second binding partnerthat binds a first partner that is attached to the host cell.Non-limiting embodiments of engineered protein and target moleculecombinations are, the engineered protein is an antibody and the targetmolecule an antigen, the engineered protein is an antigen and the targetmolecule an antibody, the engineered protein is a receptor and thetarget molecule is a ligand, the engineered protein is an enzyme and thetarget molecule is a substrate, etc., or any combination thereof.

In some embodiments the host cell is induced to express the engineeredprotein. In some embodiments, no induction step is necessary andincubating the host cell will result in the expression of the engineeredprotein. In some embodiments, engineered proteins comprising the secondbinding partner are secreted and bind to the first binding partner,thereby displaying the engineered protein on the cell surface. In someembodiments, the first binding partner is avidin and the second bindingpartner is biotin. In some embodiments, avidin is covalently conjugatedto the cell surface (e.g., directly or indirectly). Yet in someembodiments, the first binding partner is expressed by the cell anddisplayed at the host cell surface. For example, one of the bindingpartners may be expressed by the host cell as a fusion protein such as acell wall or a membrane fusion protein and displayed at the surface ofthe host cell.

Immobilization of Proteins

In some embodiments, secreted engineered proteins are immobilized on thecell surface. The invention embraces any method of immobilizing theengineered protein on the cell surface including anchoring of theengineered protein to the cell directly, e.g., if the fusion proteincomprises a cell membrane anchor (like a GPI motif), or if the fusionprotein is an integral part of a protein anchored to the membrane. Theinvention also embraces methods of immobilizing the engineered proteinson the cell surface to components that are naturally present on the cellsurface, or components that can be introduced on the cell surfacethrough overexpression. The engineered proteins can subsequently beimmobilized for instance through sulfide links (as in the case of AGA)or through linking to a sugar residue. The immobilization can bespontaneous (e.g., no change in the condition of the host cell isnecessary) or the immobilization of the engineered proteins may requirean active step, such as the addition of an agent to the host cellenvironment, or the triggering of a coupling reaction by temperature orlight. Other active coupling steps embraced by the invention are theinduction of expression, or regulation of expression, of a protein thatcan facilitate immobilization of the engineered protein to the cellsurface. Immobilization may also require the addition of a linker,spacer, or any agent that can link the engineered protein to the cellwall.

In some embodiments, the engineered proteins are immobilized through anamino acid modification. However, the invention is not so limited, andthe engineered proteins may be immobilized through any one or more aminoacids, amino acid side chains, amino acid backbone, and multiples andcombinations thereof. In some embodiments, the engineered proteins areimmobilized through an immobilization peptide. In some embodiments, theengineered proteins are immobilized and displayed at the cell surfacethrough the interaction between two binding partners having an affinityto one another, for example avidin-biotin, streptavidin-biotin,neutravidin-biotin, etc., or any combination thereof. In one aspect ofthe invention, the first binding partner and the engineered proteinfused with the second binding partners (e.g., in vivo biotinylatedproteins) are co-expressed and secreted by the host cell. Consequently,binding partners may associate intracellularly or at the cell surface.In one embodiment, the two binding partners associate intracellularlyand are exported and displayed at the cell surface as a complex. Inanother aspect of the invention, the first binding partner and theengineered protein fused with the second binding partner are secretedseparately. If the two binding partners are expressed separately, theexpression of the first binding partner may be regulated such that thefirst binding partner is expressed in sufficient amount to bind themajority of the engineered protein fused to the second binding partner,or the expression of the fusion protein is regulated such that thefusion protein is expressed in sufficient amount to be displayed at thecell surface of the secreting cell but in insufficient amount to bind toother cells in a cell population (at least not at detectable levels).

In some embodiments, the host cells are incubated in presence of asubstance slowing down the cell expression product diffusion in themedia. One should appreciate that by increasing the viscosity of theliquid medium, the protein expressed by the host cell will be morelikely to be captured by the secreting cell than by a non-secretingcell. Methods to slow down the diffusion of protein in a liquid areknown in the art and include for example PEG, gelatin etc., or anycombination thereof.

Display and Screening

Aspects of the invention provide compositions and methods for displayingmolecules (e.g., proteins) on a cell surface. In some embodiments,compositions and methods are provided for the display of high molecularweight proteins on a cell surface.

Aspects of the invention may be useful to identify one or more moleculeshaving a predetermined function of interest. By providing a system thatdisplays a cellularly expressed protein on the surface of the cell inwhich it is expressed, cells that express proteins of interest can beidentified using any assay that can be performed on a cell surface(e.g., performed on a cellular preparation to detect one or moremolecules that are displayed on the cell surface). Aspects of theinvention can be used to screen libraries expressing protein variants toidentify one or more proteins of interest. As used herein a “variant”may refer to a polypeptide or polynucleotide that differs from areference polypeptide or polynucleotide, but retains essentialproperties. A typical variant of a polypeptide differs in amino acidsequence from another reference polypeptide. Generally, differences arelimited so that the sequences of the reference polypeptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more modifications (e.g., substitutions, additions, and/ordeletions). A variant of a polypeptide may be a conservatively modifiedvariant. A substituted or inserted amino acid residue may or may not beone encoded by the genetic code (e.g., a non-natural amino acid). Avariant of a polypeptide may be naturally occurring, such as an allelicvariant, or it may be a variant that is not known to occur naturally.

Aspects of the invention provide optimized methods for identifying agenotype coding for a protein of interest. By identifying proteins ofinterest with a reporter moiety (e.g., directly or indirectly coupledwith a fluorescent moiety) or through a functional assay, cellsexpressing the protein of interest can be isolated easily. Aspects ofthe invention provide methods for identifying proteins of interestthrough the amount of protein expressed on the cell surface. Aspects ofthe invention provide opportunities for performing a broad range ofassays on cell surfaces, because of the high number and/or highconcentration of cellularly expressed molecules that can be displayed ona cell surface. Cells expressing a protein of interest may be selectedbased on their affinity to a target molecule, antigen, ligand, substrateetc., or any combination thereof. Aspects of the invention providemethods for the co-immobilization of expressed proteins and theirsubstrates or potential substrates on a cell surface. Cells expressing aprotein of interest may be isolated using Fluorescence Activated CellSorting (FACS), or any other suitable cell sorting method, or any otherhigh throughput cell screening and/or isolation method.

One aspect of the present invention provides a method for selecting hostcells displaying proteins with desirable affinity or specificity for atarget molecule (e.g., ligand or antigen) and secreting high levels ofprotein of interest. In an exemplary embodiment, in vivo or in vitrobiotinylated host cells are first incubated in the presence of solubleavidin and with a biotinylated ligand or antigen. The engineered proteinof interest having an affinity for the ligand or antigen can beexpressed in host cells. The expression of the engineered proteinresults in the immobilization of engineered proteins having an affinityfor the target molecule on the cell surface. Cells expressing theprotein of interest bound to the target molecule can be detected basedon a reporter moiety of the secreted protein or with a labeled antibodyagainst the protein of interest. Host cells displaying proteins havingdesirable affinity or selectivity for the target molecule or host cellsexpressing a desirable level of the protein of interest can be selectedbased on the intensity of the detectable label. Selected host cells maythen be incubated in absence of the biotinylated antigen and underconditions favorable for the expression and secretion of the engineeredprotein comprising a modification motif. In a preferred embodiment, themodification motif is a biotin acceptor peptide and the engineeredprotein is biotinylated in vivo. The expression of the engineeredprotein results in the immobilization of the protein on the host cellsurface through the binding of the biotinylated protein to anavidin-like protein on the cell surface. On should appreciate that ifhigh secretor host cells were selected during the first selectionprocess, host cells displaying the protein of interest on their cellsurface can then be incubated with a labeled target molecule (e.g.,epitope tag antigen or ligand). Host cells can subsequently be screenedand selected based, for example, on the affinity or specificity for atarget molecule as discussed above. Yet in another embodiment, hostcells are first selected during the first selection process fordisplaying protein with a desirable range of affinity or specificity fora target molecule, and can be subsequently screened and selected basedon the expression level of the engineered protein. The expression levelcan be determined by quantitation of a detectable label associated tothe engineered protein of interest (e.g., labeled antibody against theprotein of interest, or if the protein of interest is fused with anepitope, detectable anti-epitope antibody).

In Vitro Protein Evolution

In vitro protein evolution allows for a large number of proteinfunctions and characteristics to be investigated. In some aspects, invitro protein evolution comprises two general steps: diversification andselection. Diversification relies on the ability to generate highlydiverse libraries of nucleic acids coding for proteins. Selection can beachieved by screening the libraries for a desired phenotype and linkingthe phenotype to the genotype, e.g., by identifying the member of thelibrary that comprises the genotype that is responsible for the observedphenotype. Nucleic acid libraries can be generated through a variety ofmethods including through the introduction of mutations such as pointmutations, deletions, and insertions, or through recombination events.Methods for the generation of libraries of variants are known in the artand include error-prone PCR, synthesis of DNA in DNA repair compromisedbacteria, and chemical modification of DNA. Methods for the generationof libraries through recombination are known in the art and include geneshuffling, assembly of DNA in highly recombinogenic bacteria, syntheticnucleic acid library assembly, etc., or any combination thereof.

In some embodiments, the second step of in vitro protein evolution isselection. For a candidate protein to be selected, the variant library,or components of the variant library, and the desired functions andcharacteristics of the library members have to be evaluated. In an idealcase, each component of the library is available for evaluation. Proteinlibraries may be encoded by nucleic acid libraries, and the nucleicacids thus have to be expressed and the proteins secreted and/ordisplayed to be available for evaluation of a specific phenotype (e.g.,using an assay that interrogates cell surface or extracellularproperties). In in vitro display systems, proteins are expressed andpresented, thereby making them available for evaluation by linking themto a component of the expression system. In in vivo display systems, theproteins are expressed in an organism and displayed on the surface ofthat organism. For an overview of certain protein display technologiessee Sergeeva et al. (2006, Advanced Drug Delivery Reviews 58:1622-1654).

A library of nucleic acids can be introduced into a plurality of hostcells resulting in the expression of a member of the library in each ofthe host cells. In addition to being expressed, the proteins have to bepresented to evaluate their function or characteristic. While theproteins can be evaluated after they are secreted from the host cellinto the supernatant, it is easier to evaluate proteins if they areimmobilized. In addition, immobilization makes it easier to identify thehost cell that expresses a protein of interest. A variety of techniqueshave been developed for protein expression and display. Examples ofthese systems are ribosome display, mRNA display, DNA display, phagedisplay and cell surface display. These displays are based on theability to physically link the polypeptide produced by a library memberto its corresponding genotype.

Aspects of the invention further relate to methods and compositions forlinking a polypeptide produced by a library member for its correspondinggenotype.

Immobilizing Engineered Proteins on Cell Surfaces

In one aspect, the invention provides methods for immobilizingengineered proteins on a cell surface. In some embodiments, theengineered proteins of the invention minimally comprise a polypeptide ofinterest and an immobilization peptide. In some embodiments, theengineered protein comprises a fusion protein. In some embodiments, theengineered proteins are immobilized on the cell surface through theintroduction of an amino acid modification on the engineered protein. Insome embodiments, the amino acid modification is done in vivo (e.g.,intracellularly). In some embodiments, the modification is performedextracellularly, for instance, immediately after the engineered proteintraverses the cell membrane. In some embodiments, the amino acid to bemodified is part of a modification motif. It should be appreciated thatthe modification motif can be an integral part of the engineered proteinor it can be located on a peptide sequence that is linked to theN-terminus or C-terminus of the protein of interest, or any combinationthereof. In some embodiments, the peptide sequence that comprises themodification motif is an immobilization peptide. In some embodiments,the immobilization peptide is not directly fused to the protein ofinterest, but a spacer sequence is incorporated between theimmobilization peptide and the protein of interest. In some embodiments,a spacer sequence is a sequence of amino acid that inserted between theprotein of interest and the immobilization to allow for folding of theprotein of interest and/or modification of the immobilization peptide.In some embodiments, the spacer peptide is 5, 10, 15, 20, 50, 100 or upto 1000 amino acid residues.

In some embodiments, the engineered protein further comprises a leaderpeptide. As used herein, the term leader peptide or secretion peptide orsecretion leader peptide refers to any signaling sequence that directs asynthesized fusion protein away from the translation site, includingsignaling sequences that will result in the fusion peptide crossing thecell membrane and being secreted. The leader peptide or secretionpeptide may be proteolytically removed from the mature proteinconcomitant or immediately following export of the protein into thelumen of intracellular compartment along the secretory pathway. Theleader peptide may be a naturally occurring sequence or a syntheticsequence. In some embodiments, the leader peptide comprises themodification motif. It should be appreciated that the invention embracesany sequence order of protein of interest, immobilization peptide andleader peptide and that these elements may be separated by spacersequence, for instance an engineered protein can be characterized byleader sequence—protein of interest—spacer immobilization peptide, orprotein of interest—leader peptide—immobilization peptide. However,other sequence orders may be used as the invention is not limited inthis respect.

In some aspects of the invention, the engineered protein is immobilizedthrough the interaction with a target molecule on the cell surface. Forthe purpose of the invention, the term target molecule refers to amolecule that binds with a binding specificity to a protein such as anantibody, an antibody fragment, or an antibody-like polypeptide, areceptor, an antigen, an enzyme etc., or any combination thereof. Thetarget molecule can be for example an antigen, an epitope, a ligand, asubstrate, etc., or any combination thereof. It should be appreciatedthat the term “target molecule” can be used to refer to a substrate suchas an enzymatic substrate or a molecule that is being evaluated forbinding (e.g., a ligand, eptiope, antigen, multimerization partner suchas a homo or hetero dimeric partner, etc., or any combination thereof).Accordingly, general descriptions herein related to “target molecule”may be applied to embodiments relating to substrates and/or bindingmolecules.

In some embodiments, the target molecule is attached to the cell surfacedirectly. In some embodiments, the expressed engineered protein binds tothe target molecule, thereby displaying the engineered protein on thecell surface. However, in some embodiments where the target molecule isa substrate, the engineered protein may no bind with sufficient affinityto be immobilized or displayed on the surface of the host cell. In theseembodiments, an enzymatic reaction may be catalyzed on the cell surfaceby the engineered protein. In some embodiments, a product (e.g., adetectable product) may be immobilized on the cell surface. In someembodiments, a product (e.g., a detectable product) may be released fromthe cell surface. In some embodiments, a cell displaying a first bindingpartner at its surface binds a soluble target molecule linked to asecond binding partner thereby immobilizing the target molecule on itssurface. One should appreciate that the target or substrate moleculelinked to a second binding partner may be immobilized on the host cellsurface through binding of the second binding partner to a suitablefirst binding partner attached to the cell surface. For example, asubstrate may comprise (e.g., may be linked to) biotin and may beimmobilized on the host cell surface through its interaction with forexample avidin, Neutravidin, streptavidin or any other suitable bindingmoiety, or any combination thereof, acting as a first binding partner.The first binding partner may be directly or indirectly displayed,attached, coupled to the host cell surface. For example, avidin or anavidin-like protein may be the first binding partner and may be linkedto the cell surface by a variety of methods described herein. Forexample, biotin may be chemically coupled to the cell surface andsoluble avidin or avidin-like protein may be added extracellularly, orbiotin may be chemically attached to the cell surface via a suitablelinker and soluble avidin or avidin-like protein may be addedextracellularly, or a cell surface protein (e.g., a cell wall protein ormembrane protein) may be expressed as a biotinylated protein (e.g., viain vivo biotinylation) and avidin or an avidin-like protein may be addedextracellularly. Alternatively, a cell surface fusion protein comprisingavidin may be expressed, for instance, or avidin may be conjugated tothe cell wall and soluble biotin may be added.

It should be appreciated that the invention embraces any first bindingpartner and/or second binding partner. As used herein, binding partnersrefer to molecules that bind to each other with sufficient affinity forimmobilizing a protein or other molecule on a cell surface underconditions suitable for aspects of the invention. Although many examplesare described in the context of biotin and avidin, it should beappreciated that any suitable binding partners may be used. Forinstance, a cyclic peptide motif has been shown to bind to Neutravidinand avidin (Meyer et al., 2006, Chem. Biol. Drug Des. 68: 3-10; Gaj etal., 2007, Protein Expr. Pur. 56(1): 54-61). In another exemplaryembodiment, the first binding partner is a six-residue cyclic peptideincluding a DXaAXbPXc wherein Xa is R or L, Xb is S or T and Xc is Y orW (SEQ ID NO: 12) and the second binding partner is avidin, neutravidin,or any other suitable binding moiety. However, in some embodiments, theidentity of the first and second binding partners may be swapped asdescribed herein.

In some embodiments, the engineered protein is a fusion protein that canbind to the cell wall directly. In some embodiments, the fusion proteinis a cell surface protein. In some embodiments, the engineered proteinis expressed and displayed on the cell surface through binding to thecell surface directly. In some embodiments, the engineered protein isdisplayed on the cell surface because the cell surface protein binds tothe cell surface.

In another aspect, the invention provides for methods for displaying afirst binding partner on a cell surface by expressing a cell surfaceprotein coupled with a second binding partner and binding a firstbinding partner to the second binding partner.

Vectors

One or more engineered proteins of the invention may be encoded bynucleotide sequences. In some embodiments, the nucleotide sequences arelocated on a nucleic acid vector (e.g., containing one or moreadditional sequences useful for replication, selection, etc., or anycombination thereof). It should be appreciated that the invention coversany vector comprising nucleic acids including plasmid, phage, viruses,etc., or any other suitable nucleic acid vector. The vectors can beintroduced into a host cell prior to expression of the engineeredprotein. The vectors can be introduced by any means includingtransfection, electroporation, infection, active protein transport orthrough any other means of introducing a nucleic acid into a cell. Thevectors can be introduced immediately prior to expression, or thevectors can be introduced many cell divisions prior to expression. Thevectors can be integrated into the genome of the host cell. The vectorscan also be maintained as independent entities within the cells. Thevectors may be replicating or non-replicating vectors. The inventionalso embraces collections or libraries of vectors and libraries of cellsthat have taken up the vectors. In some embodiments, one or more nucleicacids encoding engineered protein(s) may be integrated into the genomeof a host cell without any additional vector sequences.

The vector may contain a variety of regulatory elements for maintenanceof the cell or expression of the engineered protein. Regulatory elementsinclude promoters and markers that allow for positive identification ofcells that have take up the vector. The regulatory elements may bespecies specific. Regulatory elements are known in the art and theinvention embraces any single or combination of regulatory elementsneeded or desired to express the engineered proteins.

Proteins

The invention embraces any engineered protein, protein domain, orfunctional part thereof. Engineered proteins that are particularlyembraced by the invention are engineered proteins that are functionalproteins, binding proteins, antibodies, scaffold proteins or enzymes.However, in some embodiments, an engineered protein of the invention maybe a structural protein, a storage protein, or any other protein ofinterest. A protein or enzyme of the invention may be, but is notlimited to, a therapeutic polypeptide, polymerase, ligase, restrictionenzyme, topoisomerase, kinase, phosphatase, metabolic enzyme, catalyticenzyme, therapeutic enzyme, pharmaceutical enzyme, environmental enzyme,industrial enzyme, pharmaceutical polypeptide, environmentalpolypeptide, industrial polypeptide, binding protein, antibody, antibodyfragment, single antibody chain, chimeric antibody, scaffold protein,immunotoxin, antibody-like polypeptide, signaling molecule, cytokine ora receptor. In some embodiments, an engineered protein is a polymerase.

Antibodies

In some embodiments, engineered proteins are antibodies, antibody chainsor antibody fragments. In some embodiments, the engineered proteinsinclude a protein of interest that is an antibody, antibody chain orantibody fragment. Typical antibodies have a tetrameric structure withtwo identical pairs of light and heavy chains. Both light and heavychains have, at their amino-terminus, a variable region responsible forthe specific binding to a target antigen. The carboxy-terminal region ofeach chain defines a constant region. The antibodies or fragmentsthereof may be selected for their ability to bind a specific antigen. Insome embodiments, the antibody or fragment thereof is an IgG1, IgG2,IgG3, IgG4, IgM, IgA1, IgA2, IgAsec, IgD, IgE or has an immunoglobulinconstant and/or variable domain of IgG1, IgG2, IgG3, IgG4, IgM, IgA1,IgA2, IgAsec, IgD or IgE. In other embodiments, the antibody is abispecific or multispecific antibody. In still other embodiments, theantibody is a recombinant antibody, a polyclonal antibody, a monoclonalantibody, a humanized antibody, a single chain antibody, or a chimericantibody, or a combination of two or more thereof. In some embodiments,the antibody is a human antibody. An antibody fragment of the inventionmay be, but is not limited to, a Fab fragment, a F(ab')₂ fragment, ascF_(V) fragment, a single-chain antibody, a single-domain (V_(H) orV_(L)) antibody, a camel antibody domain, a humanized camel antibodydomain, an antibody region (including one or more framework regions, oneor more constant regions, one or more variable regions, one or more CDRregions), etc., or any combination thereof.

In one aspect of the invention, the antibodies or antibody fragments areexpressed as a fusion protein comprising from the N-terminus to theC-terminus: a leader peptide (e.g., secretory signal), a first chain orfragment of a chain and a second chain or fragment of a chain (e.g.,V_(L) and V_(H); V_(L)-C_(L) and V_(H)-C_(H)). In eukaryotic cells, theleader peptide directs the fusion proteins from the endoplasmicreticulum to the Golgi apparatus to the cellular membrane. In someexemplary embodiments, the V domains of the heavy and light chains canbe expressed on the same polypeptide joined by a flexible linker (e.g.,[Gly₄-Ser]₃ linker, SEQ ID NO: 13) to form a single chain FV fragment(scFv) (see e.g., McCafferty et al., Nature, 1990, 348: 552-554).

Scaffold Proteins

In some embodiments, the engineered protein or the protein of interestis a scaffold protein. In some embodiments, the scaffold protein canbind to an antigen. In some embodiments, the engineered protein is anantibody-mimic or antibody-like polypeptide that provides a non-antibodyscaffold and one or more variable regions forming an antigen-bindingsite that interacts with the ligand or antigen molecule. Some of thepossible advantages of non-antibody scaffold include ease of selection,expression and purification, biochemical and biological properties morespecifically tailored to the physiological application and favorablepharmacokinetics. For example, a fibronectin type III domain has beenused to create an antibody-like polypeptide (see for example Parker etal., Protein Engineering Design and Selection, 2005, 18(9): 435-444).

In some embodiments, the scaffold protein is the human vitamin D-bindingprotein or a modified version of the vitamin-D binding protein. Some ofthe advantages of the vitamin D-binding protein are (1) its highconcentration in human plasma (0.5 mg/l) which suggests that the proteinis non-immunogenic; (2) its combination of high molecular weight (52 kD)and low pI (5.0) which lead to a long residence in the plasma; (3) anextensive disulfide cross-linking which provides a high thermodynamicstability; (4) the existence of a natural actin-binding site, which canbe modified to accommodate other protein binding partners; and (5) theexistence of a natural vitamin-D binding site which can be modified toaccommodate other small molecule binding partners. The vitamin D-bindingprotein binding site can be engineered to generate a library of proteinvariants that can bind a variety of target molecules.

In another embodiment, a non-antibody scaffold protein is a Cu, Znsuperoxide dismutase (SOD) or a modified version thereof. The SODprotein is an essential enzyme that protects cells from scavengingsuperoxide radical. The human form of the protein (HSOD) adopts abeta-barrel fold and forms an homodimer. Mutations at the two freecysteine residues C6A and C111S result in a thermostable version of theprotein (HSOD-AS). The HSOD-AS variant retains the wild type-fold andactivity. The advantages of the HSOD protein and the HSOD-AS variantinclude high stability, relatively small size (154 amino acids monomer)and human origin. In some embodiments, the enzymatic activity is removedthrough mutation of one or more active-site catalytic residues. In someembodiments, the designed scaffold operates as a homodimer, in someembodiments the designed scaffold is redesigned as a stable monomer. Alibrary of variants can be generated and screened for binding to aspecific target or substrate. For example, the amino acid sequence canbe varied within the loop connecting the beta-sheet elements (forexample, loops 23-28; 63-82, 102-114, 11-14; 37-40; 90-93; 120-123; and141-144).

In some embodiments the scaffold protein is human serum albumin or amodified version of human serum albumin.

Expression Systems and Host Cells

In some embodiments, the engineered proteins are expressed in vitro. Invitro expression systems are known in the art and include expressionusing cell extracts and expression systems using a mixture of proteinexpression and translation enzymes.

In some embodiments, the engineered proteins are expressed in vivo. Theengineered proteins of the invention can be expressed in any host celland the invention embraces any prokaryotic or eukaryotic cell, includingbacterial cells, yeast cells (e.g., Saccharomyces and/or Picchiaspecies), insect cells, Xenopus cells, and mammalian cells. Cells thatare particularly suited for expression of the fusion proteins of theinvention are E. Coli., S. cerevisiae, CHO and 293T cells. The cells maybe ‘wild type’ cells or the cells may be optimized for a particularcharacteristic or for a particular enzyme function that may aid inprotein expression. These cells include cells that have an optimizedcapability to take up and maintain nucleic acids, cells that haveincreased protein synthesis capability and/or cells that have increasedprotein secretion capability. Cells that can maintain the integrity ofthe nucleic acid and the synthesized proteins are particularly embraced,including cells with increased DNA repair capacity, decreasedrecombination capacity, increased protein folding capacity and/ordecreased protein degradation (e.g., protease) capacity.

In some embodiments, cells may be selected or engineered to have one ormore protease deficiencies (e.g., they lack one or more protease enzymesand/or they lack one or more protease targeting proteins) so thatexpressed proteins of interest are not degraded.

In some aspects, cells for bacterial or yeast display may be used. Insome embodiments, cell display systems feature transfection of cellswith DNA libraries and expression of library encoded polypeptides orproteins as fusions with extra-cellular receptors. Bacterial cells,yeast cells and mammalian cells can all be used for cell surfacedisplay.

It should be appreciated that any of these cellular expression and/orsecretion and/or immobilization techniques may be used in combinationwith display techniques of the invention.

Bacterial Fusion Proteins

In one aspect, the invention provides methods for the immobilization ofone or more engineered proteins, binding partners, molecular targets,substrates, etc., or any combination thereof, on a cell surface byproviding fusion proteins for display on a cell surface.

In one aspect, the invention provides methods for the immobilization ofone or more engineered proteins, binding partners, molecular targets,substrates, etc., or any combination thereof, on a cell surface byproviding fusion proteins for display on a bacterial cell surface. Insome embodiments, the bacterial fusion proteins are based on bacterialsurface proteins, which may be used for display of one or moreengineered proteins, binding partners, molecular targets, substrates,etc., or any combination thereof, on bacteria. In bacterial celldisplay, foreign gene products have been fused to surface-accessibleregions of proteins and outer membrane proteins such as OmpA, OmpC,PhoE, LamB, FhuA, and BtuB (Lang, Int. J. Med. Microbiol. 2000, 290:579-585; Etz et al., J. Bacteriol 2001, 183: 6924-6935). Insertions ofpolypeptides of more than 100 amino acids residues can be tolerated incertain cases.

Another class of bacterial surface proteins for display are transporterproteins, which contain the translocator domain mediating the outermembrane trafficking of a passenger protein. Replacement of the neutralpassenger domain of these transporters with an alternative polypeptide(e.g., one or more engineered proteins, binding partners, moleculartargets, substrates, etc., or any combination thereof) leads to thedisplay of this polypeptide on the bacterial surface in the translocatordomain. A related method for displaying proteins on the surface ofbacteria is through the use of lipoproteins, like TraT, andpeptidoglycan-associated lipoproteins (PAL), which through theirC-terminus are covalently attached to the peptidoglycan layer, but havea free N-terminus for the fusion of a polypeptide of interest (Dhillonet al., Lett. Appl. Microbiology (1999); 28: 350-354).

In one aspect, the invention provides methods for novel fusion proteinsfor bacterial surface display. A list of bacterial fusion anchoringmotifs for display of proteins of interest is provided in Table 1. Theanchoring motifs of Table 1 are selected based on their ability topresent polypeptides of interest on the bacterial cell wall.

TABLE 1 Fusion partners for bacterial surface display Fusion partnerProtein characteristics tolC, Q, R, A Membrane spanning complex OmpWOuter membrane protein, tonB, ExbB, ExbD Membrane spanning complex nfrAPhage receptor, outer membrane subunit csgG Outer membrane lipoproteinslyB Outer membrane lipoprotein yfiO Outer membrane lipoprotein SlpOuter membrane lipoprotein Blc Outer membrane lipoprotein Hyf (B, C, D,E, F) Hydrogenase subunit Frd (D, C) Fumarate reductase, membrane anchorsubunit osmC Osmotically inducible membrane protein ynfH Oxidoreductase,membrane subunit glpB Dehydrogenase, membrane anchor subunit Nuo (N, M,L, K, J, H, A) NADH::ubiquinone oxidoreductase hokC OmpX permutant Toxicmembrane protein

Yeast Surface Display

In one aspect, the invention provides methods for the immobilization ofone or more engineered proteins, binding partners, molecular targets,substrates, etc., or any combination thereof, on a cell surface byproviding fusion proteins for display of one or more engineeredproteins, binding partners, molecular targets, substrates, etc., or anycombination thereof, on a yeast cell surface. In one embodiment, theinvention provides for methods for displaying a first binding partner ona cell surface by expressing a cell surface protein coupled with asecond binding partner and binding a first binding partner to the secondbinding partner. A commonly used organism for protein display is yeast.Yeast display offers the advantage over bacteria-based technologies inthat yeast can process proteins that require endoplasmatic reticulum(ER) -specific post-translational processing for efficient folding andactivity. While mammalian cell display also facilitatespost-translational processing, yeast offers the advantage of ease ofgeneration of nucleic acid libraries, because the vectors can besimpler, and an easier introduction of the libraries into the hostcells. Most yeast expression fusion proteins are based on GPI(Glycosyl-Phosphatidyl-Inositol) anchor proteins which play importantroles in the surface expression of cell-surface proteins and areessential for the viability of the yeast. One such protein,alpha-agglutinin consists of a core subunit encoded by AGA1 and islinked through disulfide bridges to a small binding subunit encoded byAGA2. Proteins encoded by the nucleic acid library can be introduced onthe N-terminal region of AGA1 or on the C terminal or N-terminal regionof AGA2. Both fusion patterns will result in the display of thepolypeptide on the yeast cell surface.

In some embodiments, fusion proteins for yeast display include anengineered protein fused to the N-terminal or C-terminal part of aprotein capable of anchoring in a eukaryotic cell wall (e.g.,a-agglutinin, AGA1, Flo1 or major cell wall protein of lower eukaryotes,see U.S. Pat. Nos. 6,027,910 and 6,114,147 which are hereby incorporatedby reference), for example, proteins fused with the GPI fragment of Flolor to the Flol functional domain (Kondo et al., Appl. MicroBiol.Biotechn., 2004, 64: 28-40).

In addition to surface display methods based on established fusionproteins comprising a GPI anchor motif, the invention also embracesdisplay methods based on novel fusion proteins comprising a modified GPIanchor motif. Fusion proteins of the invention may comprise a protein tobe displayed (e.g., one or more engineered proteins, binding partners,molecular targets, substrates, etc., or any combination thereof), a GPIanchor and appropriate signaling sequences, which may bepost-translationally modified when the fusion protein is expressed inyeast. As a protein containing the GPI anchor and C-terminal signalingsequence is trafficked through the ER, a hydrophobic region on theC-terminal signal sequence adjacent to the GPI anchor becomes embeddedin the ER membrane, where it is cleaved by an ER protease. As the ERprotease cleaves this C-terminal signal sequence, it simultaneouslyattaches a preformed GPI anchor to the new C-terminus of the engineeredprotein (e.g., binding partner, molecular target, substrate, etc., orany combination thereof) ultimately resulting in the display of theprotein (e.g., binding partner, molecular target, substrate, etc., orany combination thereof) on the cell surface (See, e.g., Kondo et al.Appl. MicroBiol. Biotechn. 2004, 64: 28-40). The invention embracesC-terminal sequences with improved processing properties resulting inthe improved display of fusion proteins comprising the GPI-anchorproteins. Improved display comprises an increase in the number ofdisplayed proteins and/or an increase in the number of correctlyexpressed proteins. In some embodiments, C-terminal sequences withimproved processing properties are evolved by screening librariescontaining variant C-terminal sequences according to techniques known inthe art.

In one aspect, the invention provides methods for the display ofengineered proteins (one or more engineered proteins, binding partners,molecular targets, substrates, etc., or any combination thereof)comprising a fusion protein comprising a yeast display anchoring motif,wherein the anchoring motif does not comprise a GPI anchor. A yeastanchoring motif may be a cell surface protein that is partially exposedto the extracellular environment at one of its termini, and may have ahigh copy number. A protein of interest (e.g., an engineered protein,binding partner, molecular target, substrate, etc., or any combinationthereof) to be immobilized may be fused to the exposed terminus.Proteins of interest to be displayed can be fused both through their N-or C-terminus to the display fusion partner. Anchoring motifs can beexpressed with their own secretory leader sequence, or anchoring motifscan be outfitted with leader sequences that result in improvedexpression and/or secretion. A non-limiting overview of yeast anchoringmotifs is presented in Table 2.

TABLE 2 Anchoring motifs for yeast display proteins Size Copy Protein(kDa) number* Protein characteristics CCW14 23.3 42,000 Covalentlylinked cell wall protein, inner layer of cell wall CIS3 23.2 12,500Mannoprotein, internal repeat protein CWP1 24 2,060 Mannoprotein linkedthrough phosphodiester bond to beta-1,3- and beta 1,6-glucan PIR1 34.61,170 Required for cell wall stability, mediates mitochondrialtranslocation of Apn1, expression regulated by cell integrity pathwayPIR3 33 Required for cell wall stability, expression is cell cycleregulated SAG1 70 Alpha-agglutinin of alpha-cells, binds to Aga1p duringagglutination, N-terminal half is homologous to the immunoglobulinsuperfamily and contains binding site for a-agglutinin, C-terminal halfis highly glycosylated and contains GPI anchor CWP2 9 1590000 Covalentlylinked cell wall mannoprotein, major constituent of the cell wall; playsa role in stabilizing the cell wall; involved in low pH resistance;precursor is GPI-anchored STE2 48 Receptor for alpha-factor pheromone;seven transmembrane-domains STE3 53 Receptor for a-type mating factor*Copy Number based on Ghaemmaghami S, etal. (2003) Global analysis ofprotein expression in yeast. Nature 425(6959):737-41

In some embodiments, the yeast fusion proteins used for display includea cell-surface display system based on fusing an engineered protein tobe displayed to the flocculation domain of a GPI anchor protein withoutthe C-terminal portion of the GPI anchor protein (e.g., Flo1, Flo2,Flo3, Flo4, Flo5, Flo9, Flo10 or Flo11 as described in U.S. Pat. No.7,192,764 which is hereby incorporated by reference). The sugar chainsof the flocculation domain bind to the sugar chains of the cell wall,thereby displaying the engineered protein (e.g., an engineered protein,binding partner, molecular target, substrate, etc., or any combinationthereof) on the cell surface.

Carbohydrate Binding

One aspect of the invention embraces non-covalent attachment of anengineered protein (e.g., an engineered protein, binding partner,molecular target, substrate, etc., or any combination thereof) to one ormore components of the cell surface. In some embodiments, the cellsurface is the yeast cell wall. In some embodiments, the engineeredprotein comprises a protein of interest (e.g., an engineered protein,binding partner, molecular target, substrate, etc., or any combinationthereof) fused to a peptide comprising a carbohydrate binding domain. Insome embodiments, the secreted protein is attached non-covalently to thecarbohydrates present at the cell surface. In some embodiments, thesecreted protein is attached non-covalently to surface proteins on thecell surface. In some embodiments, a carbohydrate binding domain of thefusion protein may interact intracellularly with cell wall componentsbeing exported at the cell surface. In some embodiments, thecarbohydrate binding domains are fused with the C-terminus of theprotein of interest or to the N-terminus of the protein of interest orboth to the C- and the N-terminus of the protein of interest (e.g., anengineered protein, binding partner, molecular target, substrate, etc.,or any combination thereof). In some embodiments, the engineered proteincomprises multiple carbohydrate binding domains. One skilled in the artwould recognize that any carbohydrate or molecule present at the surfaceof the cell can be used to attach and immobilize the fusion protein atthe cell surface. However, it is preferable to attach the fusion protein(e.g., an engineered protein, binding partner, molecular target,substrate, etc., or any combination thereof) to molecules present inhigh copy number at the cell surface (e.g., at least 100, 1,000, 10,000copies). Carbohydrates are a major component of the yeast cell surface.Surface carbohydrates include mannoproteins, 1,3-β-glucans, 1,6-β-glucanand chitins. Mannoproteins form 30 to 50% of the cell wall mass, whereproteins account for only 4-5% of the mass and protein-linked mannosecontaining carbohydrate side chains account for the remaining mass.1,3-β-glucans compose 30-45% of the cell wall mass, forming a continuousnetwork stabilized by inter-chain hydrogen bonding. 1,6-β-glucanscompose 5-10% of the cell wall mass, forming a network with other cellwall proteins or carbohydrates. Chitin composed 1.5 to 6% of the cellwall mass, being mostly present on the intracellular side of the cellwall. Any domain known to those skilled in the art to bind molecules ofthe carbohydrate family may be fused to a protein to be immobilized atthe cell surface. For example lectins are known carbohydrate-bindingproteins or glycoproteins which are highly specific for their sugarmoieties. Examples of lectins comprise concanavalin A (ConA) which bindsinternal and non-reducing terminal alpha-mannosyl groups,phytohemagglutinin (PHA) which consists of two closely related proteinsPHA-L and PHA-E and mannose-binding lectin (MBL). Additional classes ofcarbohydrate binding domains are the carbohydrate binding modules (CBM).These modules were originally classified as cellulose binding domains(CBDs), but binding to carbohydrates other than cellulose has beenfound. CBMs are naturally found appended to catalytic modules, promotingthe association of the catalytic domain of the enzyme with itssubstrate. Examples include:

Family Protein PDB code CBM4 Laminarinase 16A (Thermotoga maritima) 1GUICBM6 Xylanase 11A (Clostridium thermocellum) 1UXX CBM9 Xylanase 10A(Thermotoga maritima) 1I8A CBM13 Xylanase 10A (Streptomycesolivaceoviridis) 1XYF CBM15 Xylanase 10C (Cellvibrio japonicus) 1GNYCBM17 Cellulase 5A (Clostridium cellulovorans) 1J83 CBM18 Agglutinin(Triticum aestivum) 1WGC CBM20 Glucoamylase (Aspergillus niger) 1AC0CBM27 Mannanase 5A (Thermotoga maritima) 1OF4 CBM29 Non-catalyticprotein 1 (Pyromyces equi) 1GWK CBM32 Sialidase 33A (Micromonosporaviridifaciens) 1EUU CBM34 α-Amylase 13A (Thermoactinomyces vulgaris)1UH2 CBM36 Xylanase 43A (Paenibacillus polymyxa) 1UX7

There are more than 300 putative CBM sequences in more than 50 differentspecies, classified into 43 families. CBMs exhibit a variety ofinteraction specificities, with different CBMs known to bind cellulose,chitin, β-1,3-glucans and β-1,3-1,4-mixed linkage glucans, xylan,mannan, galactan and starch, or lectin-like' specificity with binding toa variety of cell-surface glycans. Crystal structures are available formembers of at least 22 CBM families, including structures of 15 CBMsfrom 10 different families in complex with their oligosaccharideligands.

Known or existing carbohydrate-binding proteins, such as thoseidentified above, may be used directly as the carbohydrate bindingdomain. In some embodiments, improved carbohydrate binding domainscharacterized by a higher binding affinity, a more specific binding, ahigher stability, an increased surface expression, and/or improvedoligomeric properties are used. Further, the oligomeric structure of aknown carbohydrate-binding protein may be changed. For example, asingle-chain form of a higher-order oligomer may be created in order tomake a single polypeptide fusion of carbohydrate binding domain andprotein of interest (binding partner, molecular target etc.,).

Flo1, Flo5 and Flo10 comprise three domains: a mannose-binding domain,an intermediate repeating trans cell wall domain and a C-terminal GPIanchor signal sequence and motif (Teunissen and Steensma, 1995, Yeast,Vol. 11, pp 1001-1003). It has been demonstrated that overexpression ofthe wild-type flocculins tends to result in an increased tendency forthe cells to form flocs (Guo et al., 2001, PNAS Early Edition, (2001).However, this tendency can be somewhat mitigated by expressing atruncated version containing only the N-terminal functional domain as afusion to a target protein as has been done in earlier attempts atFLO1-mediated display systems (Matsumoto et al., 2002, Applied andEnvironmental Microbiology, 68: 9 4517-4522). In one aspect of theinvention, the N-terminal functional domain of Flol or Flol homologprotein comprising the mannose binding domain is expressed in a yeastcell. The expressed truncated protein does not contain the GPI anchormotif nor the cell wall domain but comprises the mannose binding domainallowing it to bind mannose residues in the cell wall or on the cellsurface.

In some embodiments, the carbohydrate binding domain is derived from aprotein without particular carbohydrate-binding properties. For example,the carbohydrate binding domain may be derived from a generalprotein-binding platform such as single-chain antibodies, other antibodyformats, designed ankyrin repeat proteins (DARPins), leucine-rich repeat(LRR) proteins, tetratricopeptide repeat (TPR) proteins, armadillorepeat (ARM) proteins, avimers, lipocalcins, Fn3s (including 10Fn3s, orAdNectins), linear peptides, disulfide-constrained peptides, smallmodular immunopharmaceuticals (SMIPs), tetranectins, T-cell receptors,PDZ domains, or A-domain proteins, etc., or any combination thereof.Further scaffolds include cytotoxic T-lymphocyte-associated antigen 4(CTLA-4), tendamistat, neocarzinostatin, carbohydrate-binding module 4of family 2 of xylanase from Rhodothermus marinus (CBM4-2), immunityprotein 9 (Im9), zinc finger, protein VIII of filamentous bacteriophage(p VIII), GCN4, WW domain, src homology domain 3 (SH3) domains, srchomology domain 2 (SH2) domains, TEM-1 β-lactamase, green fluorescentprotein, thioredoxin, staphylococcal nuclease, plant homeodomain finger(PHD finger), chymotrypsin inhibitor 2 (Cl2), bovine pancreatic trypsininhibitor (BPTI), Alzheimer's amyloid β-protein precursor inhibitor(APPI), human pancreatic secretory trypsin inhibitor (hPSTI), Ecotin,human lipoprotein-associated coagulation inhibitor domain 1 (LACI-D1),leech-derived trypsin inhibitor (LDTI), mustard trypsin inhibitor 2 (MTIII), scorpion toxins, insect defensin A, Ecballium elaterium trypsininhibitor II (EETI II), and cellulose binding domain (CBD).

In some embodiments, the carbohydrate binding domain is composed ofmultiple interaction domains in order to decrease the overalldissociation rate through avidity effects or to increase the bindingaffinity of the fusion protein to the carbohydrates. Each domain may beidentical, similar, or may have a different protein sequence. Eachdomain may bind the same or different carbohydrate epitopes.

Mammalian Fusion Proteins

Mammalian cells also can be used to display one or more proteins (e.g.,an engineered protein, binding partner, molecular target, substrate,etc., or any combination thereof). For example, you can use a mammaliancell protein, cell membrane protein, cell membrane binding protein, ordomain thereof to display an engineered protein (e.g., an engineeredprotein, binding partner, molecular target, substrate, etc., or anycombination thereof) on the cell surface. Mammalian cell display has theadvantage of expressing human proteins with correct post translationalmodifications such as glycosylation and phosphorylation. For instance,Chinese Hamster Ovary cell lines or derived cell lines have beensuccessfully developed to express valuable therapeutic proteins such asantibodies, and 293T cells have been used for the expression and displayof single-chain F_(v)s (Ho et al., PNAS 2006, 103: 9637-9642).

Expression

Proteins can be expressed from nucleic acids using methods known in theart. In some embodiments, protein expression is constitutive.Constitutive expression covers both expression from nucleic acids thathave been integrated in the genome and expression from nucleic acidsthat are located on non-integrated vectors. In some embodiments,expression is initiated by an activation event. Expression can beinitiated upon introduction of the nucleic acid into the cell. Theinvention particularly embraces embodiments where the protein isexpressed upon the initiation of a signal. In some embodiments, nucleicacids that encode the engineered proteins are operably connected to aninitiator sequence that regulates expression of the engineered protein.Initiator sequences that can induce expression are known in the art andinclude inducible promoters. In some embodiments protein expression isinduced. Methods of initiating protein expression are known in the artand include the addition of an activating agent (e.g., IPTG), anincrease in temperature, change in nutrient composition, change incarbon source (such as addition of sugar, methanol or glycerol), orwithdrawal of an agent from the host cell environment. In someembodiments, host cells comprising the nucleic acids encoding the fusionproteins are incubated using conditions that will result in theexpression of the protein. The conditions may be created when a cell isalready dividing, or the conditions may already be in place when thenucleic acid is introduced in the cell or when culturing of the cell isinitiated. In some embodiments protein expression is induced.

In some embodiments, protein expression occurs when the host cellcomprising a nucleic acid encoding the protein is incubated and noseparate induction step is required.

In some embodiments, two proteins are co-expressed by two expressionconstructs and secreted by the host cell simultaneously. For example,the two engineered proteins may be expressed and secreted under the sameculture conditions. In some embodiments, the two proteins are expressedsequentially. If the two engineered proteins are expressed sequentially,the host cells may be co-transfected simultaneously with two expressionconstructs under the control of two inducible promoters (e.g., twodifferent inducible promoters). Expression of the first protein isinitiated by incubating the host cells under conditions sufficient toinduce expression of the first protein and expression of the secondprotein is initiated under conditions sufficient to induce theexpression of the second protein. Alternatively, the host cells may betransfected with a first expression vector encoding a first protein andthen transfected with a second expression vector encoding a secondprotein. Alternatively, host cells may constitutively express the firstprotein and subsequently be transfected or transformed with a vectorencoding a second protein.

Display of the First Binding Partner

In some embodiments, secreted engineered proteins are bound to a firstbinding partner through a modification of the engineered protein. Insome embodiments the immobilization peptide is modified. In someembodiments, the secreted engineered proteins are bound to the firstbinding partner through a second binding partner attached to theengineered protein (e.g., immobilization peptide). In one embodiment,the first binding partner is a biotin binding partner and the secondbinding partner is biotin or biotin analog. In another embodiment, thefirst binding partner is an avidin or avidin-like binding peptide andthe second binding partner is avidin or an avidin-like protein orvariations thereof. Yet, in another embodiment, the first bindingpartner is a biotin or biotin analog and the second binding partner isavidin or an avidin-like protein or variations thereof. Avidin-likeproteins are defined as proteins that have a strong affinity for biotin.Non-limiting examples of avidin-like proteins are avidin, streptavidin,Neutravidin and modifications thereof.

In some embodiments, the secreted engineered proteins are bound to afirst binding partner, whereby the first binding partner is displayed onthe cell surface. It should be appreciated that the first bindingpartner may be attached to cell surface either in vitro or in vivo,directly or indirectly, by a variety of methods. Suitable in vitromethods include but are not limited to direct coupling to amino groupsor coupling to thiols or indirect coupling (through for example biotinor an antibody). Alternatively, the first binding partner may beexpressed and secreted by the host cell. In some embodiments, the firstbinding partner is fused to an anchoring motif and displayed at the cellsurface.

In some embodiments the first binding protein is attached to the cellsurface through a set of binding partners. In some embodiment the cellsurface displays a third binding partner. In some embodiments the firstbinding partner is linked to a fourth binding partner, which can bind tothe third binding partner, thereby displaying the first binding partneron the cell surface. In some embodiments, the third and fourth bindingpartner relate to the display of a first binding partner, targetmolecule or substrate on the cell surface. It should be appreciated thatthe third and fourth binding partners can be the same physical entitiesas the first and second binding partners. For instance, in oneembodiment of the invention both the first and a third binding partnerare biotin, while and both the second and fourth binding partner arestreptavidin. In another embodiment of the invention the first andfourth binding partner are biotin, while the second and third bindingpartner are avidin.

The display of biotin on the cell surface can be established forinstance by biotinylation of the cell surface proteins in vitro by, forexample the use of sulpho-NHS-biotin (Pierce Chemical Co.) orbiotin-sulfosuccinimidyl ester (Invitrogen). However, one shouldappreciate that as cells divide, the daughters cells do not retainbiotin. In some embodiments, biotinylation of the cell surface proteinsmay be accomplished in vivo. In some aspects of the invention, a biotinacceptor peptide (i.e., an immobilization peptide comprising amodification motif) may be fused to an anchoring cell surface protein orcell wall protein, in vivo biotinylated (e.g., with biotin ligase), andthe fusion protein comprising the surface protein or cell wall proteinand biotinylated acceptor peptide may be displayed at the cell surfaceof the host cell. In some embodiments, the cell surface protein may beengineered to include one or more biotin acceptor peptides. For example,the cell surface protein may be engineered to comprise an anchoringdomain or motif, extracellular domains and a biotin acceptor peptidefused to extracellular domains or the cell surface protein may beengineered to include more than one biotin acceptor peptide to itsextracellular terminus and to the extracellular domains. The expressionof the resulting biotinylated cell surface protein may be constitutiveor inducible. The cell surface protein is preferably a protein that isnatively localized in the cell membrane or the cell wall. Such proteinsmay be chosen based, for example, on their expression levels, theirsize, and their structural features and any combination thereof. Forinstance, preferred cell surface proteins have a high native expressionlevel. In a preferred embodiment, proteins have an extracellularN-terminus and/or C-terminus or intra-protein loops with extracellulardomains that are amenable to peptide insertion or fusion allowing thesecond (or fourth) binding partner (e.g., biotin) to freely interactwith the first (or third) binding partner at the cell surface (e.g.,avidin). In yeast, the cell surface protein includes but is not limitedto a natively present cell wall protein such as SAG1, HPS150 (PIR2P),CWP2, BIO5, SAG1, FLO5, FLO1, FIG1, FIG2, STE2, STE3, etc., or variantsof the above proteins or any other relevant cell wall proteins.

In a preferred embodiment, the engineered cell surface proteincomprising at least one biotin acceptor peptide is co-expressed with abiotin ligase, resulting in the in vivo biotinylation of the biotinacceptor peptide(s), thus displaying biotin at the host cell surface. Insome embodiments, the engineered cell surface protein comprising atleast one biotin acceptor peptide is co-expressed with a chaperoneprotein resulting in the in vivo biotinylation of the biotin acceptorpeptide(s) and display of biotin at the host cell surface. Chaperoneproteins are proteins that facilitate transport and/or folding of theengineered protein. Chaperone proteins are known in the art andnon-limiting examples include BiP, GRP94, GRP170, clanexin,calreticulin, HSP47, HSP60, HSP70, HSP90, HSP100, ERp29, ERp57, PDI andPPI. In some embodiments, the chaperone protein is BiP.

In vivo biotinylated cells can be incubated with soluble avidin at anytime point over the course of a capture assay or a screening assay. Forexample, free soluble avidin can be added at any time point during theassay thereby allowing daughters cells to be labeled with avidin oravidin-like protein and to capture biotinylated engineered proteins.Host cell culture conditions can be optimized so that the cells retainthe avidin bound to the displayed biotin. Maintaining an optimal numberof avidin on the surface of the cells displaying biotin may be achievedby, for example, incubating the cells with an increased avidinconcentration (e.g., 2 mg/ml, 5 mg/ml, 10 mg/ml, etc.), lowering thecell culture temperature (for example to 20° C. for yeast); increasingthe viscosity of the medium (e.g., by addition of PEG) or anycombination thereof.

In yet another embodiment, cell division is inhibited to maximize thepercentage of avidin-labeled cells present in the display assay. Oneshould appreciate that if after biotinylation of the cell surfaceprotein cell division is inhibited, the “parent” cells will not divideand no daughters cells are generated, and the majority of the cells willpossess avidin on their surface (see FIG. 12). Strategies for slowingcell division include, but are not limited to, temperature change,chemical treatment, or alteration of assay time scale. Chemicals used toslow cell division include, but are not limited to, hydroxyurea,nocodozole, farnesol, a-mating factor, a-mating factor, leflunomide,calcium-deprived media, EGRA, lithium, mimosine, lovastatin,aphidicolin, and thymidine.

Biotin binding moieties (e.g., avidin and avidin-like proteins) areknown in the art and include avidin and variations thereof, such asstreptavidin and neutravidin. Avidin (from egg-white), streptavidin(from Streptomyces avidinii) and Neutravidin are related proteins thatbind biotin with similar dissociation constants of about 10⁻¹⁵ M (Green,1975, Adv. Protein Chem., vol. 29, pp. 85-133). NeutrAvidin protein is adeglycosylated version of avidin and binds to a lesser extent tolectins. Avidin and avidin like proteins are constructed of fournon-covalently attached identical subunits, each of which bears a singlebiotin-binding site and exhibit the same three dimensional fold. Eachprotein functions as a homo-tetramer, where four identical copies of themonomer associate into the native quaternary structure, each monomerbinding one molecule of biotin. Two monomers associate to form a primaryor structural dimer, two of which then combine to form a tetramer (forreview, see e.g., Laitinen et al., Trends Biotech. , Vol. 25, No8, pp269-277, 2007). The unique feature of the binding of avidin to biotin isthe strength and specificity of formation of the avidin-biotin complex.The resultant affinity constant, estimated at 1.6×10⁻¹⁵ M for avidin and2.5×10⁻¹³ M for streptavidin, is the highest known for a protein and anorganic ligand. The strong affinity of avidin or streptavidin to biotinis dependent on the tetrameric configuration of the protein. Avidinmonomer shows a highly reduced affinity constant (10⁻⁷ M, Laitinen etal., 2003, J. Biol. Chem. Vol. 278, pp 4010-4014). In some embodiments,avidin is expressed by the cell together with the engineered protein tobe displayed. Recombinant forms of avidin and streptavidin have beenengineered and produced in eukaryotic and prokaryotic expression systemswith modified properties of charge, oligomerization and ligandspecificity.

It should be appreciated that the methods described herein may beimplemented with any avidin, avidin-variant or avidin-like proteindescribed herein.

Oligomeric proteins have been successfully displayed at the cell surfaceby expressing one subunit fused with an anchoring motif and secretingthe remaining subunits. For example, hetero-oligomeric functionalantibody's Fab fragment has been successfully displayed on the yeastcell surface by expressing the light chain Fab fragment as a fusionFab-α-agglutinin protein and a fragment of the Fab heavy chain as asecretion protein (Lin Y. et al., 2003, Appl. Microbiol. Biotechnol.,Vol. 62, pp 226-236). Moreover, Furukawa et al., were able to displaystreptavidin on a yeast cell surface by co-expressing native subunitsand anchored subunits fused with the C-terminus of 318 amino acids ofFlo1p (2006, Biotechnol. Prog., Vol. 22, pp 994-997). In someembodiments, an avidin monomer fused with an anchoring motif isdisplayed on the cell-surface. In some embodiments, additional copies ofavidin are expressed and secreted by the host-cell. In some embodiments,the displayed avidin monomer fused with an anchoring motif binds toadditional avidin proteins secreted by the host cell.

It should be appreciated that avidin and avidin-like proteins can alsobe expressed as two polypeptide dimer chains (Nordlund et al., 2004, J.Biol. Chem., Vol. 279, pp36715-36719 and WO05047317) or as a singletetrameric polypeptide chain (Nordlund et al., Biochem. J., 2005, Vol.392, pp 485-491). In one embodiment, avidin or avidin-like protein isexpressed as two single chain avidin dimers wherein one single chaindimer is expressed as a protein fused to an anchoring motif and thesecond single chain dimer is expressed and secreted by the host cell. Inanother embodiment, avidin or avidin-like proteins are expressed andsecreted as single chain tetramers fused to an anchoring motif. In yetother embodiments, the different subunits of the avidin or avidin likeprotein are co-expressed and secreted as a single-chain dimeric avidin,a monomeric avidin and a monomeric avidin subunit fused to anchoringmotif. In another embodiment, avidin is displayed at the surface of thehost cell as a fusion of a monomeric or dimeric avidin-anchoring motifprotein. Anchoring motifs include, but are not limited to, any knowncell wall proteins (e.g., a-agglutinin, Flolp), GPI anchors, modifiedGPI anchors, AGA2, etc., or any other anchoring motif described hereinor known to one of skill in the art. Engineered tetrameric, dimeric, andmonomeric avidin may be design to bind biotin with an affinityrepresented by a dissociation constant of about 10⁻⁷M, about 10⁻⁸ M,about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M, about 10⁻¹² M, about 10⁻¹³M, about 10⁻¹⁴ M or about 10⁻¹⁵ M.

In some embodiments, a first binding partner such as avidin,streptavidin or avidin-like protein is conjugated (also referred to asconnected) to cell surface directly (See FIG. 2). The first bindingpartner can be covalently bound to the cell surface or the first bindingpartner can be bound to the cell surface through other bindinginteractions, including binding interactions based on affinity. Thecovalent conjugation of the first binding partner to the cell surfacecan be accomplished using a variety of connectors. A connector is anymolecule that can covalently conjugate the binding moiety to the cellsurface. In some embodiments, a hetero-bifunctional connector is used.Non-limiting examples of heterobifunctional connectors areC6-succinimidyl 4-hydrazinonicotinate acetone hydrazone (C6-SANH),succinimidyl 4-hydrazinonicotinate acetone hydrazone (SANH),succinimidyl 4-hydrazidoterephthalate hydrochloride (SHTH), succinimidyl4-formylbenzoate (SFB) and C6-succinimidyl 4-formylbenzoate (C6-SFB). Insome embodiments, the connector comprises N-hydroxysuccinimide (NHS;which can react with free amines) and aldehyde-reactive hydrazidefunctional groups. Non-limiting examples of connectors comprising NHSand hydrazide groups are C6-SANH and SANH.

A non-limiting example of a hetero-bifunctional connector that can beused to conjugate a first binding partner comprising free amines, likeavidin, or a carbohydrate containing binding partner, to the cellsurface, is C6-succinimidyl 4-hydrazinonicotinate acetone hydrazone(C6-SANH). The first binding partner can be conjugated to the cellsurface by labeling the binding partner with C6-SANH. This conjugationoccurs by reacting the NHS groups with free amines on the surface of thefirst binding partner (if the binding moiety is a polypeptide, solventexposed lysines may provide the free amines). In a subsequent step, thehydrazide moiety may be reacted with the free aldehydes on the cellsurface, thereby covalently conjugating the first binding partner to thecell surface. In some embodiments, the cells are pre-treated withperiodate to generate free aldehydes on the carbohydrates of the cellsurface. Alternatively, if the first binding partner comprises acarbohydrate group, the conjugation can be performed by reacting thecells with C6-SANH to form covalent bonds between free amines on thecell surfaces and the NHS ester moiety of the connector. The cells canthen be mixed with periodate-treated binding moiety using theconnector's free hydrazide group to create a stable connection betweenoxidized carbohydrate on the first binding partner and the cell surface.However, it should be appreciated that any other suitable technique maybe used to connect the first binding partner to the cell surface as theinvention is not limited in this respect.

In some embodiments, a binding partner is connected to the cell surfacethrough a spacer. In some embodiments, the first binding partner isconnected to the cell surface through a spacer. The first bindingpartner can be covalently bound to the spacer or the first bindingpartner can be bound to the spacer through other binding interactions,including binding interactions based on affinity. In some embodiments,the spacer comprises a biotin moiety and is referred to as a biotinspacer. In some embodiments, the first binding partner is a biotinbinding moiety, such as avidin, and the spacer comprises biotin and alinker element. In some embodiments, the linker is attached to the cellsurface. In some embodiments a spacer comprises a set of bindingproteins. In some embodiments the first binding partner is bound to afourth binding partner, which is connected to a third binding partner,which is attached to the cell surface.

Spacers are not limited to interactions between binding partners and theinvention embraces any moieties that can function as a spacer betweenthe cell wall and the first binding partner. In some embodiments,spacers, including the biotin spacer, can be of any length and compriseany kind of linker, including, alkanes, PEG, etc., or any combinationthereof. In some embodiments the linker is attached to the cell surface,through the action of cell wall binding protein. Preferably the spaceris chemically inert and does not react with any other component of thehost cell, engineered protein or agent of the cellular environment ofthe host cell. Molecules that are suitable for linkers in spacers areknown in the art. A spacer may be connected to the cell surface and theinvention embraces the linking of the spacer to any cell surface moiety,including sugars, amino acids and lipids. In some embodiments, thespacer is linked to an amino acid side chain (e.g., through the actionof an N-succimidyl ester).

In some embodiments, the secreted engineered protein and the spacer arebound to the same biotin binding moiety. In some embodiments, thesecreted engineered protein is bound through its biotin to avidin, andthe avidin is also bound to the biotin moiety of the biotin spacer,thereby immobilizing the engineered protein to the cell surface. In someembodiments, more than one engineered protein is bound to one avidin.

Modification Motif

In some embodiments the coupling of the second binding partner to themodification motif comprises an amino acid modification. In someembodiments, “an amino acid modification” comprises the result of anymodifying event that results in the modification of the amino acid,including modifications of the peptide backbone and modifications of theamino acid side chain. In some embodiments, the amino acid side chain isoxidized, reduced or cross-linked. In some embodiments, an agent iscoupled to the amino acid. In some embodiments, modification of an aminoacid comprises coupling of a second binding partner. In someembodiments, the second binding partner is coupled to the amino acidmodification motif by a coupling enzyme. In some embodiments, themodification is produced intracellularly. For example, the amino acidmodification may be a post-translational modification of the engineeredprotein. Aspects of the invention also incorporate secondarymodification, for example, modification of a first amino acid or aminoacid sequence which in turn induces modification of a second amino acidor amino acid sequence of the fusion protein.

In some embodiments, a modification motif is a sequence of amino acidsthat directs a certain modification event. In some embodiments, amodification motif is a sequence of amino acids that acts as a substratefor a certain modification event. Modification motifs also may besequences that can undergo a chemical reaction, for instance, thesequence may comprise one or more cysteines, which can form di-sulfurbridges, one or more aromatic rings, which can form cross-links, or oneor more side chains that can participate in chemical reactions. In someembodiments, the sequence can function as a substrate for an enzymaticmodification event. Non-limiting examples of enzymatic modificationevents are phosphorylation, glycosylation, ubiquitination, acetylation,or other side-chain modifying event. Enzymatic modifications also may bethe coupling of peptides, proteins or other biomolecules likecholesterol, and coenzymes like biotin, or other biomolecules.

Coupling of the second binding partner also may involve the addition ofnon-biomolecules (e.g., molecules that are not naturally present in thecell). The invention embraces both one-step and multi-stepmodifications. In some embodiments, a first modification is produced,which can undergo a second modification, resulting ion the coupling ofthe second binding partner. In some embodiments, the second bindingpartner is coupled extracellularly.

It should be appreciated that the modification motif may be created bymutation a protein of interest, or fusing a peptide sequence thatincludes a modification motif to the protein of interest.

In Vivo Biotinylation

In some embodiments, the modification motif may be a sequence thatundergoes coupling of a second binding partner. In a preferredembodiment, the modification motif is a biotin acceptor peptide and thebinding partner is biotin. One should appreciate that modification canbe produced intracellularly or extracellularly. For example, anengineered protein comprising a biotin acceptor peptide may be expressedin a host cell and secreted at the host cell surface at its cellsurface. Biotin may then be supplied extracellularly in the culturemedium.

In one aspect the invention provides methods for the in vivobiotinylation of engineered proteins. Biotin is an essential coenzymesynthesized by plants, most bacteria and some fungi. Biotin isbiologically active only when protein-bound and intracellular biotin iscovalently attached to a class of metabolic enzymes, the biotincarboxylases and decarboxylases. These enzymes catalyze the transfer ofCO₂ to and between metabolites, by use of the biotin cofactor as amobile carboxyl carrier, and are key enzymes of gluconeogenesis,lipogenesis, amino acid degradation and energy transduction. Biotinprotein ligase (BPL) is an enzyme responsible for attaching biotin tobiotin carboxylases and decarboxylases. BPL catalyses thepost-translational formation of an amide-linkage between the carboxylgroup of biotin and the ε-amino group of a specific lysine residue ofthe carboxylase and decarboxylase (Chapman-Smith et al., Biomol.Engineering, 1999, 16: 119-125). The invention covers any enzyme thatcatalyzes coupling of biotin to a protein or polypeptide. In someembodiments, the coupling enzyme is a biotin protein ligase (BPL). Insome embodiments, the BPL is BirA (the E. Coli BPL). Bir A polypeptidesand nucleic acids encoding BirA are described in U.S. Pat. No. 6,255,075and U.S. Pat. No. 5,723,584 which are hereby incorporated by reference.A humanized version of BirA is also known in the art (J. Biotechnol.2005, 0: 245-249). Sequences of BirA and BPL proteins are shown in Table3. However, all BPLs are covered by the invention including BPL from S.cerevisiae (Cronan et al. FEMS Kett 1995, 130: 221) and human BPL(Suzuki et al. Nat. Genetics 1994, 8: 122-128). The invention covers theuse of any BPL, including modified and mutated forms of BPL, in any hostcell (e.g., the use of E. Coli BPL (BirA) in S. cerevisiae ). In someembodiments, BPL is a temperature sensitive BPL, which can be activatedby lowering or increasing the temperature to a specific level. BPLrequires both ATP and biotin to couple biotin to a polypeptide. In someembodiments, host cells are grown under conditions sufficient forcoupling biotin to a polypeptide. In some embodiments, the conditionsare a high enough concentration of ATP and a high enough concentrationof biotin. In some embodiments, ATP and/or biotin are added to the hostcell environment to initiate or accelerate coupling of biotin to apolypeptide. Each BPL has a natural polypeptide substrate sequence towhich biotin is coupled (the biotinylation sequence) and the inventionembraces using biotinylation sequences from any BPL, syntheticbiotinylation sequences or any combination thereof. Examples of BPLsubstrate sequences can be found in Chapman et al. (Biomol. Engineering,1999, 16: 119-125). Examples of commercially available BPL substratepeptides are the Bioease™ Tag of Invitrogen, which comprises a 72 aminoacid peptide derived from Klebsiella pneumoniae, AviTag™ of Avidity™,which comprises a peptide of 15 amino acids and PinPoint™ of Promegawhich comprises a 128 amino acid peptide. Biotinylation sequences arealso described in U.S. Pat. No. 5,723,584, U.S. Pat. No. 5,252,466, U.S.Pat. No. 5,874,239, U.S. Pat. No. 6,265,552 which are herebyincorporated by reference. Cell that overexpress BirA and plasmidscoding for BirA are commercially available from Avidity (Denver, Colo.)and include E. Coli strains AVB 99, AVB 100 and AVB 101. In someembodiments, the modification motif comprises a biotinylation sequence.A peptide comprising a biotinylation motif is also referred to as abiotinylation peptide or a biotin acceptor peptide. In some embodiments,the modification motif comprises a biotinylation peptide and the aminoacid modification comprises coupling biotin to the biotinylationpeptide.

In some embodiments, the coupling enzyme is expressed simultaneouslywith the engineered protein. In some embodiments, the coupling enzyme isexpressed from a vector. In some embodiments, the gene coding for thecoupling enzyme is integrated into the genome of the host cell. In someembodiments, the gene for the coupling enzyme is located on the samevector as the gene for the engineered protein.

It should be appreciated that the biotinylation methods may be used tobiotinylate any protein of interest or cell bound protein describedherein.

TABLE 3 Sequences of BirA and BPL proteins  SEQ ID NO: 1 E. coli BirAWT MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK  SEQ ID NO: 2 BirAv1 MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRQVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK  SEQ ID NO: 3 BirAv2 MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDACIAEYQQAGRGRQGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRQVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK  SEQ ID NO: 4 E. coli BirAWTS MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDASIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK  SEQ ID NO: 5 BirAv1S MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDASIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRQVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK  SEQ ID NO: 6 BirAv2S MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRDWGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSGDASIAEYQQAGRGRQGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRKLGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRQVEESVVNQGWITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIGDKEIFGISRGIDKQGALLL EQDGIIKPWMGGEISLRSAEKSEQ ID NO: 7 S. cerevisiae BPL1 WT MNVLVYNGPGTTPGSVKHAVESLRDFLEPYYAVSTVNVKVLQTEPWMSKTSAVVFPGGADLPYVQACQPIISRLKHFVSKQGGVFIGFCAGGYFGTSRVEFAQGDPTMEVSGSRDLRFFPGTSRGPAYNGFQYNSEAGARAVKLNLPDGSQFSTYFNGGAVFVDADKFDNVEILATYAEHPDVPSSDSGKGQSENPAAVVLCTVGRGKVLLTGPHPEFNVRFMRKSTDKHFLETVVENLKAQEIMRLKFMRTVLTKTGLNCNNDFNYVRAPNLTPLFMASAPNKRNYLQEMENNLAHHGMHANNVELCSELNAETDSFQFYRGYRASYDAASSSLLHKEPDEVPKTVIFPGVDEDIPPFQYTPNFDMKEYFKYLNVQNTIGSLLLYGEVVTSTSTILNNNKSLLSSIPESTLLHVGTIQVSGRGRGGNTWINPKGVCASTAVVTMPLQSPVTNRNISVVFVQYLSMLAYCKAILSYAPGFSDIPVRIKWPNDLYALSPTYYKRKNLKLVNTGFEHTKLPLGDIEPAYLKISGLLVNTHFINNKYCLLLGCGINLTSDGPTTSLQTWIDILNEERQQLHLDLLPAIKAEKLQALYMNNLEVILKQFINYGAAEILPSYYELWLHSNQIVTLPDHGNTQAMITGITEDYGLLIAKELVSGSSTQFTGNVYNLQPDGNTFDIFKSLIAKKVQSSEQ ID NO: 8 S. cerevisiae BPL1 variant 1 (KR to KK) MNVLVYNGPGTTPGSVKHAVESLRDFLEPYYAVSTVNVKVLQTEPWMSKTSAVVFPGGADLPYVQACQPIISRLKHFVSKQGGVFIGFCAGGYFGTSRVEFAQGDPTMEVSGSRDLRFFPGTSRGPAYNGFQYNSEAGARAVKLNLPDGSQFSTYFNGGAVFVDADKFDNVEILATYAEHPDVPSSDSGKGQSENPAAVVLCTVGRGKVLLTGPHPEFNVRFMRKSTDKHFLETVVENLKAQEIMRLKFMRTVLTKTGLNCNNDFNYVRAPNLTPLFMASAPNKKNYLQEMENNLAHHGMHANNVELCSELNAETDSFQFYRGYRASYDAASSSLLHKEPDEVPKTVIFPGVDEDIPPFQYTPNFDMKEYFKYLNVQNTIGSLLLYGEVVTSTSTILNNNKSLLSSIPESTLLHVGTIQVSGRGRGGNTWINPKGVCASTAVVTMPLQSPVTNRNISVVFVQYLSMLAYCKAILSYAPGFSDIPVRIKWPNDLYALSPTYYKKKNLKLVNTGFEHTKLPLGDIEPAYLKISGLLVNTHFINNKYCLLLGCGINLTSDGPTTSLQTWIDILNEERQQLHLDLLPAIKAEKLQALYMNNLEVILKQFINYGAAEILPSYYELWLHSNQIVTLPDHGNTQAMITGITEDYGLLIAKELVSGSSTQFTGNVYNLQPDGNTFDIFKSLIAKKVQSSEQ ID NO: 9 S. cerevisiae BPL1 variant 2 (KR to KK, aglyco) MNVLVYNGPGTTPGSVKHAVESLRDFLEPYYAVSTVNVKVLQTEPWMSKTSAVVFPGGADLPYVQACQPIISRLKHFVSKQGGVFIGFCAGGYFGTSRVEFAQGDPTMEVSGSRDLRFFPGTSRGPAYNGFQYNSEAGARAVKLNLPDGSQFSTYFNGGAVFVDADKFDNVEILATYAEHPDVPSSDSGKGQSENPAAVVLCTVGRGKVLLTGPHPEFNVRFMRKSTDKHFLETVVENLKAQEIMRLKFMRTVLTKTGLNCNNDFNYVRAPSLTPLFMASAPNKKNYLQEMENNLAHHGMHANNVELCSELNAETDSFQFYRGYRASYDAASSSLLHKEPDEVPKTVIFPGVDEDIPPFQYTPNFDMKEYFKYLNVQNTIGSLLLYGEVVTSTSTILNNNKALLSSIPESTLLHVGTIQVSGRGRGGNTWINPKGVCASTAVVTMPLQSPVTNRATSVVFVQYLSMLAYCKAILSYAPGFSDIPVRIKWPNDLYALSPTYYKKKNLKLVNTGFEHTKLPLGDIEPAYLKISGLLVNTHFINNKYCLLLGCGISLTSDGPTTSLQTWIDILNEERQQLHLDLLPAIKAEKLQALYMNNLEVILKQFINYGAAEILPSYYELWLHSNQIVTLPDHGNTQAMITGITEDYGLLIAKELVSGSSTQFTGNVYNLQPDGNTFDIFKSLIAKKVQSSEQ ID NO: 10 S. cerevisiae BPL1 variant 3 (aglyco) MNVLVYNGPGTTPGSVKHAVESLRDFLEPYYAVSTVNVKVLQTEPWMSKTSAVVFPGGADLPYVQACQPIISRLKHFVSKQGGVFIGFCAGGYFGTSRVEFAQGDPTMEVSGSRDLRFFPGTSRGPAYNGFQYNSEAGARAVKLNLPDGSQFSTYFNGGAVFVDADKFDNVEILATYAEHPDVPSSDSGKGQSENPAAVVLCTVGRGKVLLTGPHPEFNVRFMRKSTDKHFLETVVENLKAQEIMRLKFMRTVLTKTGLNCNNDFNYVRAPSLTPLFMASAPNKRNYLQEMENNLAHHGMHANNVELCSELNAETDSFQFYRGYRASYDAASSSLLHKEPDEVPKTVIFPGVDEDIPPFQYTPNFDMKEYFKYLNVQNTIGSLLLYGEVVTSTSTILNNNKALLSSIPESTLLHVGTIQVSGRGRGGNTWINPKGVCASTAVVTMPLQSPVTNRATSVVFVQYLSMLAYCKAILSYAPGFSDIPVRIKWPNDLYALSPTYYKRKNLKLVNTGFEHTKLPLGDIEPAYLKISGLLVNTHFINNKYCLLLGCGISLTSDGPTTSLQTWIDILNEERQQLHLDLLPAIKAEKLQALYMNNLEVILKQFINYGAAEILPSYYELWLHSNQIVTLPDHGNTQAMITGITEDYGLLIAKELVSGSSTQFTGNVYNLQPDGNTFDIFKSLIAKKVQSSEQ ID NO: 11, Streptococcus pneumonia birA MKSYQAVYQILSKETDYISGEKIAEKLSLSRTSIWKAIKRLEQEGIEIDSIKNRGYKLMNGDLILPEILEENLPIKVSFKPETKSTQLDAKEAIDLGHEANTLYLASYQTAGRGRFQRSFYSPQGGIYMTLHLKPNLPYDKLPSYTLLVAGAVYKAIKNLTLIDVDIKWVNDIYLNNHKIGGILTEAMTSVETGLVTDIIIGVGINFTIKDFPQELKEKAASLFKATAPITRNELIIEIWRAFFETPAEELLYLYKKQSFILGKEVTFTLEQKDYKGLAKDISENGKLLVQCDNGKEIWL NSGEISLNSWK 

Secretion

In some embodiments, an engineered protein comprising a modificationmotif and a secretion leader peptide is secreted from the host cell. Insome embodiments, cells are grown under conditions that result insecretion of the engineered fusion proteins. Secretion may occur withoutfurther induction and may be a continuous process started by theinduction of expression of the protein, or secretion may be induced bychanging one or more conditions in the cellular environment, independentfrom the induction of expression. In some embodiments, secretion of theengineered proteins is directed by a secretion peptide that is a part ofthe engineered protein. In addition, secretion may be facilitated bychaperone proteins (e.g., PDI, BiP etc.), that confer better folding ofthe engineered protein or protein complex, reduce aggregation propensityto the engineered protein (into for example non-functional structures)or confer better secretory trafficking, proteins that can transport theengineered proteins, or through proteins that can facilitate vesicleformation. In some embodiments, vectors comprising a nucleic acidencoding the chaperone proteins are transfected into the host cell underthe control of the constitutive or inducible promoter. In someembodiment, the host cells are over-expressing chaperone proteins. Insome embodiments, the nucleic acid encoding the chaperone proteins areintegrated in the genome of the host cell. Other proteins that play arole in protein secretion may also be used (e.g., expressed along with aprotein of interest). It should be appreciated that one or morechaperone proteins or other proteins may be encoded on and expressedfrom the genome of the host cell. In some embodiments, the host cellsare optimized for secretion. For example, cell lines with increasedexpression of proteins that aid in secretion of the engineered proteinsmay be used, and host cells with optimized cell membranecharacteristics, such as permeability, facilitating crossing of themembrane by the engineered proteins may be used.

In some embodiments, a first binding partner or an engineered proteincomprising a first binding partner and an engineered protein comprisinga second binding partner are co-expressed in a host cell and secreted bythe host cell simultaneously. For example, the two engineered proteinsmay be expressed and secreted under the same culture conditions. If thetwo engineered proteins are expressed sequentially, the host cells maybe co-transfected simultaneously with two expression constructs, oneexpression construct comprising the nucleic acid sequences encoding afirst binding partner or a first engineered protein comprising a firstbinding partner under the control of a first constitutive or induciblepromoter and a second expression construct comprising nucleic acidsequences encoding a second engineered protein comprising a secondbinding partner under the control of a second inducible promoter, thefirst binding partner being constitutively expressed or expressed afterinduction of the first promoter at the surface of the cell, the secondbinding partner being expressed and secreted by the cell after inductionof the second promoter.

Alternatively, the host cells may be transfected with a vector encodingan engineered protein comprising a first binding partner underconditions such that the first binding partner is expressed at thesurface of the host cells. The cultured cells may then be transfectedwith a vector encoding a second engineered protein comprising a secondbinding partner under conditions such that the second binding partner isexpressed and secreted by the host cells. Alternatively, host cells mayconstitutively express an engineered protein comprising a first bindingpartner and subsequently a vector encoding the engineered proteincomprising a second binding partner is introduced into the host cells.

In another aspect of the invention, the host cells may be transfectedwith a vector encoding an engineered cell surface protein comprising asecond binding partner and incubated under conditions that the secondbinding partner is expressed at the surface of the host cells. Thecultured cells may then be incubated with a soluble first bindingpartner, resulting in the display of the first binding partner. Thecells may subsequently be transfected with a vector encoding anengineered protein, to which a second binding partner can be coupled invivo or an engineered protein comprising a second binding partner underconditions such that the engineered protein comprising the secondbinding partner is expressed and secreted by the host cells.

In another embodiment, host cells may constitutively express theengineered cell surface protein comprising the second binding partnerand/or the engineered protein of interest comprising the second bindingpartner. Alternatively, the host cells may express constitutively or maybe induced to express a soluble first binding partner (e.g., avidin).The first binding partner may be directly or indirectly attached orcoupled to the host cell surface as described above. For example, thefirst binding partner (e.g., avidin) may be linked to the cell surfacevia a second binding partner (e.g., biotin) that has been chemicallycoupled to the cell surface or chemically attached to the cell surfacevia a suitable linker. Host cells may be co-transfected or transfectedsubsequently with a vector encoding an engineered protein comprising asecond binding partner under the control of a constitutive or induciblepromoter.

It should be appreciated that the proteins described herein also may beencoded on the genome of the host cell in addition to, or instead of,the vectors.

Display of Target Molecule

In some embodiments a target molecule is expressed on the cell surface.In some embodiment an engineered protein is expressed with affinity forthe target molecule. The expressed engineered protein can bind to thetarget molecule resulting in the display of the engineered protein.

In another aspect of the invention, host cells may be co-transfectedwith a vector encoding an engineered cell surface protein comprising afirst binding partner and a vector encoding an engineered protein ofinterest having an affinity for a target molecule. The cultured cellsmay then be incubated under conditions that the first binding partner isexpressed at the surface of the host cells. Cells displaying the firstbinding partner are incubated in presence of a soluble target moleculecoupled with a second binding partner, wherein binding of the secondbinding partner to the first binding partner results in display of thetarget molecule at the surface. The cells may then be incubated underconditions that the engineered protein of interest is expressed andsecreted by the cell and under conditions favorable for the binding ofthe engineered protein of interest to the target molecule displayed atthe surface of the host cells. Alternatively, the host cells mayconstitutively express the engineered cell surface protein comprisingthe second binding partner and/or the engineered protein of interest.

In some embodiments a soluble second binding partner is added to cellsdisplaying a third binding partner, resulting in binding of the secondbinding partner and display of the second binding partner. It should beappreciated that the third binding partner can be a moiety similar orthe same as a first binding partner, and a cell displaying a thirdbinding partner an be generated in the same way as a cell displaying athird binding partner is generated. Once a cell displaying a thirdbinding partner is generated a soluble second binding partner can beadded resulting in the display of the second binding partner. As a nextstep a target molecule attached to a first binding partner can be addedresulting in the display of the target molecule.

In some embodiments, the first or third binding partner is avidin,neutravidin, streptavidin, avidin-like protein or any biotin-bindingprotein and the second binding partner is biotin or avidin-bindingpeptide.

In some embodiments, host cells may be transfected with a vectorencoding a first binding protein (e.g., avidin), a vector encoding anengineered protein of interest having an affinity for a target moleculeand a vector encoding a construct comprising a target molecule and asecond binding partner. One should appreciate that in this manner allthree of the principle components may be expressed, processed, andsecreted in vivo. The three components may be expressed in a host celland secreted by the host cell simultaneously or sequentially, resultingin the display of the target molecule and the binding of the engineeredprotein to the target molecule.

Reporter Moiety

In some embodiments, an engineered protein comprises a reporter moiety.In some embodiments, an engineered protein comprises a first reportermoiety and/or a target molecule comprises a different second reportermoiety. The reporter moiety can be N-terminally or C-terminally linkedto the protein of interest of the engineered protein and/or the targetmolecule, or the reporter moiety can be linked to the immobilizationpeptide and/or the secretion peptide. The invention embraces anyconfiguration of an operably linked engineered protein comprising animmobilization peptide (comprising the immobilization motif) andoptionally a secretion peptide, and optionally a reporter moiety, or anycombination thereof. In some embodiments, the reporter moiety is afluorescent protein.

Fluorescent proteins are known in the art and include Green FluorescentProtein (GFP) and color variants thereof like YFP (Yellow FluorescentProtein) and DsRed. Reporter moieties also include proteins orpolypeptides that can process a substrate that can readily be detectedby an assay. For example, proteins in this group include peroxidases.Reporter moieties further include polypeptides that can be detected bybinding the polypeptide to a labeled antibody, including the FLAG®peptide and the His6 affinity tag. The reporter moieties allow for theselection of cells that have secreted an engineered protein. In someembodiments, FACS (Fluorescence Activated Cell Sorting) can be used toidentify and isolate host cells that secrete an engineered protein.However, other fluorescence based techniques may be used (e.g., afluorescence-aware colony picker, for example available from Genetix).

Screening of Antigen or Ligand Binding Proteins

Some aspects of the invention are related to the display and screeningof proteins that bind to a target protein, such as an antigen or aligand. Some aspects of the invention are related to the display andscreening of antigen binding proteins including antibodies, antibodyfragments and scaffold proteins. In some embodiments, the antigenbinding proteins are displayed on the host cell surface using any of thecell display methods of the invention. In some embodiments the antigenbinding proteins are coupled to a second binding partner and displayedon the cell surface by binding to a first binding partner. In someembodiments, the antigen binding fragments are displayed by binding to atarget molecule (e.g., an antigen) bound to the cell surface. In someembodiments, the antigen binding proteins including, antibodies,antibodies fragments, antibodies chains or scaffold proteins areexpressed as fusion proteins comprising a secretion leader peptide, abiotin acceptor peptide (comprising the modifications motif) andoptionally a FLAG epitope. The biotin acceptor peptide can be fused atthe N-terminus of the protein and the Flag epitope can be fused to theC-terminus of the protein or the biotinylation acceptor peptide can befused to the N-terminus of the protein and the Flag epitope to theC-terminus of the protein.

In some embodiments, genes encoding the antibody heavy and light chainsare under the control of the same promoter. In some embodiments, genesencoding the antibody heavy and light chains are under the control ofdifferent promoters. For example, genes encoding the heavy and lightchains may be cloned respectively under the control of GAL1 and GAL10 orGAL10 and GAL1 promoters in opposite direction for expression of theantibodies in yeast. Yeast expression vectors are known in the art andare commercially available. Exemplary vectors include the pESC vectorsfrom Stratagene.

In one aspect, libraries of promoters are provided to optimize the ratioof light and heavy chain expression. In some embodiments, the librariescomprise mutations within the promoter's consensus sequence. Ineukaryotic cells, the TATA box (or Goldberg-Hogness box) is a DNAsequence found in the promoter region of most genes. The TATA box has acore 5′-TATAAA-3′ DNA sequence. Sequence analysis reveals that thenucleotide at the second position, e.g., nucleotide A, is highlyconserved in yeast. In some embodiments, a mutated-TATA box library ofNANNNN sequences is generated. In some embodiments, a mutated-TATA boxlibrary of NANNNN sequences is generated for each one of the promoterscontrolling the heavy chain and the light chain expression. As anexample, when one position is conserved and five positions arenon-conserved, the combinatorial library includes 10⁵ TATA box sequencesfor each promoter (e.g., NANNNN). In some embodiments, a library of TATAsequences is generated by random mutagenesis. In some embodiments, thelibrary is screened to identify the TATA boxes variants having a desiredproperty. For example, the TATA box library may be screened to identifythe TATA boxes having increased antibody or antibody fragment expressionover a wild type TATA box. Expression of the antibody or antibodyfragment under the control of TATA box variants may be increased atleast 2 times, at least 5 times, at least 10 times compared to theexpression of the antibody or antibody fragment under the control of thewild type TATA box.

Screening of Host Cells

In one aspect of the invention, the host cells displaying the secretedengineered protein may be screened and selected for the expression levelof the engineered protein, the stability of the engineered proteinand/or the affinity to a target molecule of the engineered protein.

Some aspects of the invention relate to methods for screening for cellsexpressing high levels of a protein of interest. In an exemplaryembodiment, in vivo or in vitro biotinylated cells are incubated withsoluble avidin and under conditions to allow secretion of thebiotinylated engineered protein comprising a protein of interest. Cellsthat display the engineered protein are detected by labeling theengineered protein. In some embodiments the cells that display theengineered protein are detected by binding to a labeled antibody againstthe protein of interest or using a detectable anti-class antibody. Insome embodiments, the protein of interest is fused with an epitope-tag(e.g., FLAG peptide from Sigma-Aldrich), and the cells secreting theprotein of interest may be labeled using a detectable anti-epitopeantibody (for example a monoclonal anti-FLAG antibody). Selection ofcells expressing high levels of protein of interest can be carried outusing multiple rounds of cell sorting and amplification by cell culturegrowth. Each round of selection involves the sorting of cells on thebasis of the intensity of a detectable label such as fluorescence.Separation may be done by any of the methods known in the art includingthe fluorescence activated cell sorting (FACS) system, the magnetic cellsorting system (MACS), or any other suitable cell separation or sortingtechnique.

Some aspects of the invention relate to methods to screen for cellsexpressing a protein of interest (e.g., antibody, antibody-mimicproteins, scaffold proteins, receptors) that can interact with aspecific target molecule (e.g., antigen or ligand) with a desiredspecificity. Other aspects of the invention relate to the enrichment fora protein of interest having a high (e.g., highest or optimized)specificity for a target molecule (e.g., antibodies having a high, forexample highest or optimized, affinity for an antigen).

In an exemplary embodiment, in vivo or in vitro biotinylated host cellsare first incubated in the presence of soluble avidin and with abiotinylated ligand or antigen. In some embodiments, the ligand orantigen is labeled using an epitope-tag (e.g., His6 tag). The cellsdisplaying the antigen at their surface are then incubated to allowsecretion of a protein of interest (e.g., receptor, antibody, enzyme,scaffold protein or any other protein of interest) having an affinityfor the ligand or antigen. Cells expressing the protein of interestbound to the antigen can be detected based on the reporter moiety of thesecreted protein, the ligand or both, and can be isolated by variousmethods. In some embodiments the secreted proteins or ligand are notbiotinylated. In some embodiments, cells are labeled with a labeledantibody against the protein of interest or with a detectable anti-classantibody to detect the protein of interest and with a labeled antibodyagainst the antigen or with antibody recognizing a tag epitope on theantigen. Alternatively, if the protein of interest is fused with anepitope-tag (e.g., the FLAG peptide from Sigma Aldrich), the cellssecreting the protein of interest may be labeled using a detectableanti-epitope antibody (for example a monoclonal anti-FLAG antibody).

Selection of host cells displaying, for example, a high affinity orspecificity (e.g., extraordinarily high affinity or specificity) for theligand or antigen of host cells secreting high levels of protein ofinterest may be carried out using multiple rounds of cell sorting andamplification by cell culture growth. Each round of selection involvesthe sorting of cells on the basis of the detectable label such asfluorescence. In some embodiments, libraries of candidate proteins arescreened for their ability to bind a surface displayed antigen. A rangeof ligand or antigen concentration may be used for different rounds ofsorting depending on the desired affinity of a displayed protein for itsligand or antigen. Separation may be done by any of the methods known inthe art including the fluorescence activated cell sorting (FACS) system,the magnetic cell sorting system (MACS), or any other suitable cellseparation or sorting technique.

It should be appreciated that techniques described herein may be usedfor sorting, screening, selecting, and/or isolating cells on the basisof two or more properties (e.g., two or more of expression, display,affinity, activity, etc.). In some embodiments, cells that are isolatedon the basis of one or more properties may be further evaluated (e.g.,the encoded engineered proteins may be assayed for one or more functionsor properties of interest). For example, cells may be isolated based onthe level of expression and/or binding properties and/or enzymaticproperties of displayed engineered protein variants encoded by thecells. The encoded proteins subsequently may be assayed (e.g., in thecontext of a cellular display system, or after isolation and/orpurification from the host cell) for one or more similar or additionalproperties (e.g., in an in vitro assay, in a non-cellular environment,and/or when administered as a research or pharmaceutical preparation toa subject such as a mammal, e.g., a human).

Evaluation of Engineered Proteins

In one aspect, the invention provides methods for evaluating if anengineered protein has a predetermined function or property. In oneaspect, the invention provides methods for assaying for a predeterminedfunction or property of an engineered protein. It should be appreciatedthat property and function can be used interchangeably and include anamount and/or level of any of the following: protein expression,secretion, display, enzymatic activity, binding affinity (e.g., antigenand/or ligand binding), stability, etc., or any combination thereof.Accordingly, a predetermined function or property may be any physical,chemical or biological characteristic of a protein, including but notlimited to, stability, size, structure, resistance towards proteases,enzymatic properties including substrate specificity, binding propertiesincluding antigen or ligand binding etc., or any combination thereof.Predetermined protein functions are protein functions that may bedesired for a specific protein, e.g., increased stability or increasedresistance to proteases. In addition, a predetermined function may beevaluated through comparison to a threshold level of the functionality.In some embodiments, proteins with a certain level of predeterminedfunctionality or property may be selected. In some embodiments the levelof engineered protein that is displayed is assayed by detecting a levelof a reporter molecule, epitope tag, antigen, or ligand that is attachedto the engineered protein. In some embodiments the protein property canbe evaluated by challenging the protein to a specific condition. Forinstance, if the predetermined property is protein stability, proteinsabove a certain resistance towards a chaotropic reagent can be selected.The selected proteins may subsequently be analyzed. Methods of analysismay include the determination of the primary and secondary sequence. Insome embodiments, the selected proteins may be pooled and subjected toone or more rounds of selection. In some embodiments, the selectedproteins may be subjected to one or more additional structural orfunctional assays.

To evaluate whether an engineered protein has a predetermined function,the engineered protein can be expressed in a host cell. In someembodiments, expression is induced by the addition of an agent to theenvironment of the host-cell. The expression of the engineered proteinresults in the immobilization of the engineered protein on the cellsurface through binding of the engineered protein to a first bindingpartner. Any of the embodiments described herein can be used to arriveat an engineered protein bound to a first binding partner or targetmolecule, wherein the first binding partner or target molecule isconnected to the cell surface, thereby resulting in the immobilizationof the engineered polypeptide. Once immobilized, the engineered proteincan be evaluated for the predetermined protein function.

In some embodiments, libraries of nucleic acids encoding protein ofinterest variants can be expressed and screened for the predeterminedfunction. In some embodiments, libraries of cells comprising nucleicacids encoding the protein of interest variants can be expressed andscreened for the predetermined function or property. It should beappreciated that libraries of nucleic acids encoding the protein ofinterest variants can be screened for multiple properties or functions.In addition, the libraries can be subjected to multiple rounds ofscreening for one or more properties resulting in an enrichment of thelibrary for that one or more properties.

Methods for evaluating protein functions are known in the art. In someembodiments, the engineered protein will comprise a reporter moiety,such as a fluorescent moiety, and the signal of the reporter moiety canbe used to evaluate a specific protein function. The specific assay usedto evaluate a protein function depends on the particular proteinfunction being evaluated. For instance, if the predetermined function isprotein stability, an immobilized engineered protein can be challengedwith chaotropic reagents or to increased temperature, and changes in thephysical structure of the protein (e.g., protein folding) can beobserved, wherein a higher stability is correlated with the capacity tomaintain protein folding at higher chaotropic concentrations.

Stability of a protein or a protein variant may be critical for thefunction of expressed proteins (e.g., single chain antibody, see e.g.,Worn et al., J. Biol. Chem., 2000, 275: 2795-2803). Standard approachesto evaluate the stability of a protein include measuring the meltingtemperature of the protein (T_(m)) using for example scanningcalorimetry and/or the free energy of unfolding (ΛG) at a specifictemperature (e.g., 25° C.) using, for example, guanidium hydrochlorideor urea denaturation followed by tryptophan intrinsic tryptophanfluorescence or circular dichroism. Thermal stability has been shown tobe correlated to the secretion and the yeast cell surface display ofsingle chain T cell receptor (Shusta et al. J. Mol. Biol., 1999, 292:949-956 and U.S. Pat. No. 6,300,065). In some embodiments, a measure ofthe expression levels of the proteins or protein variants may be used toselect for more stable proteins. According to aspects of the invention,more stable proteins have higher expression levels and can be identifiedby isolating highly expressed proteins (e.g., proteins that areexpressed at higher levels than an initial or reference protein). Insome embodiments, the expression level is determined by labeling theprotein using a reporter moiety to determine the amount of expressedprotein per cell or per cell surface area. Populations of cellsexpressing a more stable variant may be identified and isolated byfluorescence activated cell sorting (FACS). The highest expressingpopulations may be collected by FACS and subjected to a subsequent roundof sorting, thereby enriching the population with cells expressing themore stable protein variant. In some embodiments, a library of proteinvariants may be screened for protein expression to select for proteinvariants with improved biophysical properties (e.g., stability). In oneembodiment, protein variants showing an expression level of at least 5,10, 20, 40 or 50 fold higher than the corresponding wild type proteinare selected.

If the desired predetermined function to be evaluated is insensitivitytowards proteases, the immobilized protein can be challenged withincreasing concentrations of one or more proteases, and the integrity ofthe engineered protein monitored. If the predetermined function is abiological function, such as a specific enzymatic activity, an assayappropriate to that particular enzymatic function can be performed.Enzymatic assays are performed routinely art and a person of ordinaryskill in the art will know what enzymatic assay to use to evaluate aspecific enzymatic function.

Evaluation of Protein Complexes

In one aspect, the invention provides methods for evaluating whether aprotein complex has a predetermined function or property. In one aspect,the invention provides methods for assaying the predetermined functionof a protein complex. A protein complex comprises two or more engineeredproteins that interact with each other. To evaluate whether a proteincomplex has a predetermined function, a protein complex comprising oneor more engineered proteins is produced and evaluated for apredetermined function (e.g., one or more engineered proteins may beexpressed under conditions that allow them to interact and form aprotein complex that can be evaluated in vivo or in vitro). Engineeredproteins of a protein complex can interact with each other when one ormore of the engineered proteins are expressed and immobilized on a hostcell surface. Embodiments of methods for expressing and immobilizingengineered proteins on a cell surface have been described above. In someembodiments, the immobilized protein complex can be evaluated for apredetermined function. Techniques that are used to evaluate apredetermined function of a protein complex may be the same techniquesas those that are used to evaluate embodiments of engineered proteins.

It should be appreciated that the engineered proteins of the proteincomplex do not have to be processed in the same way. For instance afirst engineered protein of a protein complex can be expressed from avector which is transiently present in a host cell, while the secondengineered protein of the protein complex is expressed from a geneintegrated in the genome of the cell. Two engineered proteins can beexpressed simultaneously, or a first protein can be expressed andimmobilized on the cell surface prior to expression and immobilizationof a second engineered protein. In some embodiments, a first engineeredprotein is immobilized on the cell surface and a second protein is addedto the environment of the host cell, after which it interacts with thefirst engineered protein to form a protein complex. In some embodiments,one of the engineered proteins of the protein complex is a component ofa library while the second engineered protein is not part of a library.This last embodiment allows for the evaluation of the components of alibrary of first engineered protein for the ability to form a proteincomplex or to have a particular function or level of activity with asecond engineered protein. It should be appreciated that in someembodiments, only one protein of a protein complex is engineered to besecreted and anchored to a cell surface. The presence of at least onemember of a protein complex anchored on a cell surface may be used toimmobilize other members of the protein complex (e.g., engineered ornon-engineered proteins) that interact with the anchored protein.

It should be appreciated that techniques described herein may be usedfor evaluating, sorting, screening, selecting, and/or isolating proteinsor protein complexes on the basis of two or more properties (e.g., twoor more of expression, display, affinity, activity, etc.).

It should be appreciated that any of the assays for cellular, protein,protein complex, and/or substrate analysis described herein may be basedon standard binding, detection, and/or enzymatic assays. For example,display levels may be based on detecting a reporter molecule (e.g., anantigen and/or eptitope, an enzymatic reporter that produces adetectable product, a functional reporter, or any other detectable orselectable reporter that allows quantification and/or selection based onpredetermined levels of expression and/or activity, or any combinationthereof).

Evaluation of Substrate Processing by an Engineered Protein

In one aspect, the invention provides methods for evaluating whether anengineered protein can process a substrate. In one aspect, the inventionprovides methods for assaying if an engineered protein can process asubstrate. In another aspect, the invention provides methods forselecting an engineered protein capable of processing a substrate with adesired processing activity. In some embodiments, engineered proteinsare screened for specific enzymatic activity. Enzymes of interestinclude, but are not limited to, polymerase, ligase, restriction enzyme,topoisomerase, kinase, phosphatase, metabolic enzyme, industrial enzymesetc, or any combination thereof. Processing a substrate may involve themodification of a substrate by an engineered protein or an interactionof the engineered protein with the substrate. Any substrate is embracedby the invention including polypeptides, nucleic acids, lipids,polysaccharides, synthetic polymers or synthetic compounds. Processing asubstrate or interacting with a substrate may involve, but is notlimited to one or more of the following processes: binding to thesubstrate, dissociating from the substrate, nicking the substrate,cutting the substrate, activating the substrate, deactivating thesubstrate, charging the substrate, decharging the substrate, changingsubstrate conformation, copying the substrate, replicating thesubstrate, conjugating molecules to the substrate, conjugating peptidesto the substrate or modifying the substrate. In one embodiment, toevaluate whether an engineered protein can process a substrate, theengineered protein is expressed in a host cell, as described herein,resulting in the immobilization of the engineered protein on the cellsurface through binding of the engineered protein to a binding partner.Immobilized engineered proteins can subsequently be evaluated for theability to process a substrate. The particular assay used to evaluatewhether an engineered protein can process a substrate will depend on thespecific processing event and/or substrate being evaluated. Assays toevaluate whether an engineered protein can process a substrate are knownto people of ordinary skill in the art. The assays of the invention canbe used to screen libraries of nucleic aids or libraries of host cellscomprising nucleic acids encoding variants of proteins of interest.

Substrate Coupled to Cell Surface

In some embodiments, the substrate is coupled to a cell surface. Anymethod for coupling the substrate to a cell surface is embraced by theinvention. Coupling the substrate to a cell surface may comprise thedirect coupling of the substrate to the cell surface (e.g., to thesurface carbohydrates or surface protein) or coupling the substrate tocell surface may comprise coupling the substrate to a linker which isconnected to the cell surface. In some embodiments, the substrate iscoupled to a binding partner that is connected to the cell surface. Insome embodiments, the substrate binding partner is the same substratebinding partner to which the secreted engineered polypeptide is bound.In some embodiments, the substrate comprises a biotin moiety and thebinding partner is a biotin binding partner, such as avidin. In someembodiments, the biotin binding moiety is bound to a biotin spacer,thereby connecting the biotin binding moiety to the cell surface. Insome embodiments, the biotin spacer, the substrate comprising biotin andthe engineered polypeptide are all bound to the same biotin bindingagent. In some embodiments, the substrate and the engineered polypeptideare bound to different biotin binding agents. In some embodiments,multiple substrates and/or multiple engineered polypeptides are bound toan immobilized binding partner. Immobilizing both the substrate and theengineered protein may bring them into close proximity, thereby allowingfor optimized assays to evaluate whether an engineered protein canprocess a substrate. However, it should be appreciated that theengineered proteins can still be evaluated for their ability to processa substrate even if the substrate is not coupled to the cell surface.For instance, the ability to process a substrate can be evaluated byadding the substrate to the host-cell environment.

In some embodiments, the processing of a substrate generates adetectable signal or a change in the level or type of a signal. Thenature of the signal will depend on the specific assay used to evaluateprocessing of the substrate. In some embodiments, the signal is afluorescent signal. Fluorescent signals can be generated through avariety of processing methods. Non-limiting examples of the generationof fluorescent signals are the incorporation in, or coupling of, afluorescent moiety to a substrate. The invention also embraces assaysbased on the disappearance of a fluorescent signal, for instance theremoval of a fluorescent moiety from a substrate by processing of thesubstrate, and assays based on a change in fluorescent signal. Assaysbased on a change in fluorescent signal also include FRET (FluorescenceResonance Transfer), where a change in fluorescent signal is dependenton a change in distance between two fluorescent moieties. Assays basedon a fluorescent signal also cover assays based on the generation of afluorescent signal through a secondary event. For instance, afluorescently labeled antibody can be added to a substrate that is beingprocessed to monitor for the appearance/disappearance of a particularsubstrate characteristic.

Evaluation of Substrate Processing by a Protein Complex

In one aspect, the invention provides methods for evaluating whether aprotein complex can process a substrate. In one aspect, the inventionprovides methods for assaying if a protein complex can process asubstrate. A protein complex comprises one or more engineered proteinsthat interact with each other. In some embodiments, the engineeredproteins of the protein complex are expressed in the host cell andimmobilized on the host cell surface, resulting in an interactionbetween the engineered proteins of the protein complex. Embodiments forevaluating whether a protein complex can process a substrate are similarto embodiments for evaluating whether an engineered protein can processa substrate, as described herein. In some embodiments, the substrate iscoupled to the cell surface.

Screening of Candidate Engineered Proteins

In one aspect, the invention provides methods for screening candidateengineered proteins. In some embodiments, the method comprises theintroduction of a library or plurality of vectors into a population ofhost cells. In some embodiment, the population of host cells displays afirst binding partner at its cell surface. Each vector may comprise agene coding for a unique engineered protein and components to allow forthe expression of the gene in the host cell. In addition, eachengineered protein may comprise a modification motif. In someembodiments, the modification motif is the same for each engineeredprotein. The host cells are grown under conditions sufficient to induceexpression of the engineered proteins and produce a modification on themodification motif. In some embodiments, the modification motif is animmobilization peptide and the modification comprises coupling of asecond binding partner to the modification motif. The modifiedengineered proteins are secreted and bind to a first binding partnerwhich is connected to the cell surface in vivo or in vitro. In someembodiments, the first binding partner is avidin and the second bindingpartner is biotin. In yet other embodiments, the first binding partneris biotin and the second binding partner is avidin. Once the engineeredproteins are immobilized on the cell surface the proteins can beevaluated for a predetermined function. In some embodiments, the membersof the library are compared to each other or to an engineered proteinwith a known predetermined function.

In some embodiments, the predetermined function comprises the processingof a substrate. In some embodiments, processing of a substrate resultsin a signal (e.g., change in fluorescence, change in color, or loss ofsuch a signal originally incorporated in the unprocessed substrate,etc.). In some embodiments, the signal generated by processing of thesubstrate is compared to a threshold level. Comparing the signal levelto a threshold level facilitates the identification of engineeredproteins with a predetermined function. For instance if the thresholdlevel of a signal for a particular substrate processing event is 10, andthe engineered proteins are evaluated for the ability in that particularprocessing event, then any engineered protein with a signal higher than10 is a candidate engineered protein. In contrast, if a desired proteinfunction is to have less activity for a certain process (for instance,an unwanted non-specific side reaction) than an engineered protein witha signal lower than 10 is a candidate engineered protein. In someembodiments, the threshold signal is a signal generated by a polypeptidewith random coil structure. In some embodiments, the threshold signal isa signal generated by a wild type version of the engineered candidateprotein. In some embodiments, the threshold signal is a signal generatedby a commercially available variant of the engineered protein.

It should be appreciated that any of the binding or substrate assaysdescribed herein may be used to identify or select protein or targetmolecule variants that have one or more new or modified properties orfunctions. For example, polymerases with increased processivity, lowererror rates, increased salt and/or thermal stability, etc., or anycombination thereof may be identified and/or isolated according toaspects of the invention.

Libraries

It should be appreciated that any of the protein functions or propertiesdescribed herein may be evaluated, screened for (or against) or selected(or against) in the context of a single type of cell expressing a singletype of engineered protein or in the context of a library of cells eachexpressing a different engineered protein or protein variant. In oneaspect, the invention provides methods for generating libraries ofengineered proteins or libraries of nucleic acid and/or polypeptidecomponents, such as vectors of nucleic acid coding for the engineeredproteins that can be used to generate libraries of engineered proteins.In some embodiments, a library comprises two or more variants of anengineered protein or component thereof, e.g., two or more variants ofan engineered protein wherein each variant comprises a unique engineeredprotein with only a minor change in amino acid composition. In someembodiments, a library comprises two or more unrelated sequences. Forinstance, to identify a candidate engineered protein that can inhibit anenzyme, a library of engineered proteins with random sequence orpre-determined sequences may be interrogated. A library can have atleast 2, at least 5, at least 10, at least 50, at least 100, at least1000, at least 10,000, at least 100,000, at least 1,000,000, at least10⁷, at least 10⁸, at least 10⁹, at least 10¹⁰ or at least 10¹¹ members.

Libraries of the invention include libraries of host cells, wherein eachhost cell expresses a unique engineered protein and wherein eachengineered protein comprises a unique polypeptide linked to animmobilization peptide (e.g., the same immobilization peptide). Thevectors can be integrated into the genome of the host cells or thevectors can be freely replicating, e.g., plasmids that have beenintroduced into the host cell but have not been integrated in the genomeof the host cell. The library can also comprise a combination of hostcells comprising freely replicating and integrated vectors. Libraries ofthe invention also may be libraries of host cells, wherein each hostcell displays on its surface a unique fusion protein, and wherein eachengineered protein comprises a unique polypeptide coupled to animmobilization peptide.

In some embodiments, the library provides host cells with a high densityof engineered proteins immobilized on the cell surface. In someembodiments, the high density is accomplished by binding multipleengineered polypeptides to one binding partner. In some embodiments, thenumber of engineered proteins per cell is greater than 10³, greater than10⁴, greater than 10⁵, greater than 10⁶, greater than 10⁷, or greaterthan 10⁸ engineered proteins per cell. In some embodiments, theimmobilization peptide is a biotinylation peptide. In some embodiments,the immobilization peptide is a transmembrane protein. In someembodiments, the immobilization peptide comprises a GPI anchor. In someembodiments, the immobilization peptide is a peptide that is naturallypresent on the cell surface. In some embodiments, the immobilizationpeptide is a peptide that binds one or more molecules naturally presenton the cell surface (e.g., surface carbohydrates or proteins on the cellsurface).

The invention also embraces libraries of vectors, wherein each vectorcomprises a nucleic acid encoding a unique engineered protein andwherein each engineered protein comprises a unique polypeptide coupledto an immobilization peptide and/or comprises a mobilization motif

The invention also embraces libraries of engineered proteins, whereineach engineered protein comprises a unique polypeptide coupled to animmobilization peptide.

In any of the libraries of the invention, the engineered protein cancomprise a therapeutic polypeptide, polymerase, ligase, restrictionenzyme, topoisomerase, kinase, phosphatase, metabolic enzyme, catalyticenzyme, therapeutic enzyme, pharmaceutical enzyme, environmental enzyme,industrial enzyme, pharmaceutical polypeptide, environmentalpolypeptide, industrial polypeptide, binding protein, antibody, antibodyfragment, signaling molecule, cytokine, receptor, or any combination oftwo or more thereof.

In some embodiments, libraries of antibodies or other binding proteins(e.g., single chain antibodies, scaffold proteins, etc.) may beevaluated or screened to identify and/or isolate variants that i) bindto a chosen antigen and/or epitope and/or other target molecule (e.g., anovel antigen and/or epitope) and/or ii) have high (e.g., increased)affinity for a particular antigen and/or epitope and/or other targetmolecule. Methods of the invention may be designed to identifyantibodies or other binding proteins that have affinities, for aparticular antigen and/or epitope and/or other target molecule, greaterthan a binding affinity represented by a dissociation constant of about10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about 10⁻¹¹ M, about10⁻¹² M, about 10⁻¹³ M, about 10⁻¹⁴ M or about 10⁻¹⁵ M. In someembodiments, a single antibody or other binding protein may be assayedto against a library of target molecules to identify one or more targetpeptide sequences (e.g., novel or modified ligand, antigen, epitope,receptor, dimerization, mulitmerization, or other binding motifs orproteins) that bind to the antibody or other binding protein. Similarly,methods of the invention may be designed to identify target peptidesequences that have affinities, for a particular antibody or otherbinding protein, greater than a binding affinity represented by adissociation constant of about 10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about10⁻¹⁰ M, about 10⁻¹¹ M, about 10⁻¹² M, about 10⁻¹³ M, about 10⁻¹⁴ M orabout 10⁻¹⁵ M.

Production of Proteins

In order to be able to produce proteins of interest, the nucleic acid ofthe selected host cell is traditionally recovered, amplified and clonedinto an expression vector. For example, the DNA of a selected antibody,or protein of interest producing cell can be extracted from the hostcell, amplified, cloned, and expressed to produce proteins with desiredantigen specificity or other characteristic.

One aspect of the invention relates to the display, screening andproduction of a protein of interest in a host cell. In one embodiment,the host cell displaying a protein of interest is selected and may beswitched from a displaying mode to a producing mode. In a display mode,a library of proteins variants is expressed by a population of hostcells and immobilized at the cell surface. As described above, thedisplay mode allows for the screening of a library of proteins variantsand sorting of the proteins variants based on their expression level,stability or affinity for a target molecule. In the production mode,cells expressing protein variants that have been selected in the displaymode, secrete the protein of interest in the extracellular medium. Oneshould appreciate that depending on the mode of display, omitting onestep involving the expression/ display/ addition of one of the bindingpartner of the display system can lead to the secretion of the proteinof interest from the cell. For example, if a protein of interest needsto undergo in vivo biotinylation to be displayed at the cell surface,non-expression of the BirA gene (by repression, non-induction etc. . . )will result in the expression of non-biotinylated cell wall proteins orthe expression of non-biotinylated protein of interest and will lead tothe secretion of the protein of interest. If the display system involvesthe step of binding avidin to the biotin on the cell surface, incubationof the cells in an avidin-free medium will result in the secretion ofthe protein of interest. Alternatively, if the host cells surfaceproteins are chemically biotinylated in vitro, incubating the cells in abiotin free medium will also result in the secretion of the protein ofinterest. Any method of switching the cell from display mode toproduction mode is embraced by the invention.

Aspects of the invention are illustrated by the attached figures thatrelate to non-limiting embodiments of expression and display systems.For example, FIGS. 1-2 illustrate embodiments of an engineered proteinthat is biotinylated in vivo, secreted, and displayed on the cellsurface by binding to avidin attached to the cell surface. FIGS. 3-4provide experimental results using non-limiting display methods of theinvention. FIGS. 5-6 illustrate additional non-limiting examples ofprotein modification and display using methods and compositions of theinvention. FIGS. 7-12 illustrate non-limiting examples of antibodydisplay applications using compositions and methods of the invention.FIGS. 13-16 illustrate non-limiting examples and experimental results ofsubstrate display assays using compositions and methods of theinvention. These and other aspects of the present invention are furtherillustrated by the following Examples, which in no way should beconstrued as further limiting. The entire contents of all of thereferences (including literature references, issued patents, publishedpatent applications, and co-pending patent applications) citedthroughout this application are hereby expressly incorporated byreference.

EXAMPLES Example 1 Protein Immobilization Through IntracellularBiotinylation

In vivo protein display methods typically rely on the expression ofproteins as fusions to cell wall, cell membrane, or phage particleproteins. As an alternative to these traditional methods, proteins maybe expressed from vectors and biotinylated in vivo. The biotinylatedproteins may be subsequently secreted or excreted into the supernatantwhere the biotinylated proteins may bind to avidin that has beenattached to the cell surface via a biotin linker. This “biotin-avidinsandwich” immobilizes the biotinylated proteins on the surface of thecell. Proteins immobilized on cells are fluorescently labeled andphenotypic assays are performed to select proteins with desirablephenotypes. Cells with immobilized proteins with desirable phenotypesare isolated by flow cytometry or another form of selection such aspanning against immobilized antigen. If desired, multiple rounds ofselection and isolation are performed. The isolated cells are expandedin culture and the nucleic acid coding for the protein with thedesirable phenotype is identified. Optionally, an N-terminal secretionsignal is used for expression in eukaryotic cells. The protein istranscribed and subsequently biotinylated through the overexpression ofthe BirA biotinylating enzyme. In the next step, the protein is secretedinto the supernatant or excreted through mild cell surfacepermeabilization performed by media supplement addition or theco-expression of permeabilizing proteins. Finally, the biotinylatedprotein is immobilized on the surface of the cell by avidin, conjugatedto the cell surface directly or anchored to the cell surface through abiotin-PEG-NHS linker or biotinylated cell wall protein. A synopsis ofan example of a biotinylation protein engineering system is presented inFIG. 1 and FIG. 2.

In vivo Biotinylation

The method for capturing biotinylated protein on the cell surface can becombined with the method of biotinylating and secreting the protein ofinterest. Protein biotinylation in vitro is a common molecular biologytechnique, and can also be performed in vivo (Samols, D., Thornton, C.G., Murtiff, V. L., Kumar, G. K., Haase, F. C., and Wood, H. G. (1998)J. Biol. Chem, 263, 6461-6464). Most of the proteins that are naturallybiotinylated in vivo act as biotin transporters. One such transporter isthe biotin carboxyl carrier protein (BCCP) in E. coli. BCCP isbiotinylated on lysine side chains by an enzyme called biotin haloenzymesynthetase (BirA) (Fall, R. R. (1979) Methods in Enzymology, 62,390-398; Barker, D. F. and Campbell, A. M. (1981) J. Molecular Biology,146, 469-492; Howard, P. K. Shaw, J., and Otsuka, A. J. (1985) Gene, 35,321-331; Barker, D. F., and Campbell, A. M., (1981b) J. MolecularBiology, 146, 451-467). In vivo biotinylation of heterologous proteins(i.e., proteins that are not natural substrates for biotinylation) isaccomplished by fusing a BCCP domain or a BCCP domain mimic to theprotein of interest and expressing the fusion protein in conjunctionwith overexpression of the BirA gene (Cronan, J. E. (1990) J. ofBiological Chemistry, 265, 10327-10333; Yamano, N., Kawata, Y., Kojima,H. Yoda, K., and Yamasaki, M. (1992) Bioscience, Biotechnology, andBiochemistry, 56, 1017-1026; Schatz, P. J. (1993) BioTechnology, 11,1138-1143; Tsao, K. L., DeBarbieri, B., Michel, H. and Waugh, D. S.(1996) Gene, 169, 59-64). An interesting in vivo biotinylation system isthe BIOTRX construct (Smith, P. A., Tripp, B. C., DiBlasio-Smith, E. A.,Lu, Z., LaVallie, E. R., McCoy, J. M. (1998) Nucleic Acids Research,36(6), 1414-1420). This construct comprises a small biotin acceptorpeptide fused to the N-terminal region of thioredoxin. Thioredoxin is asmall protein normally involved in cytosol redox but that can also beused as a fusion partner for heterologous protein production. The BIOTRXconstruct correctly directs the production of biotinylated IL-12 invivo.

Secretion/Excretion of Biotinylated Protein

The in vivo biotinylation system is designed to excrete or secrete thebiotinylated proteins into the supernatant without irreparably damagingthe cell. For eukaryotic systems, such as yeast, the biotinylatedprotein is expressed and secreted by fusing the protein to an N-terminalsecretion signal sequence. Numerous examples of signal sequences existfor S. cerevisiae and any one of them can be used to direct secretion.Because the secretory pathway is spatially segregated from the cytosolin eukaryotes, the BirA gene also needs to be directed to the secretorypathway through the N-terminal addition of a secretion signal. The BirAprotein can be co-secreted with the engineered protein or retained inthe secretory pathway through the C-terminal addition of the HDELendoplasmic reticulum retrieval sequence. Prokaryotes such as E. colilack an advanced secretory apparatus. The delivery of the biotinylatedprotein into the supernatant therefore may require adjusted growthconditions or specialized cell lines. Adding supplements, such asglycine, or detergents, such as Triton X-100, to the cell environmentcan promote cell membrane permeability allowing highly expressedproteins to leave the cell via mildly compromised cell membranes (Jang,K. H., Seo, K. B., Song, K. B., Kim, C. H., Rhee, S. K., (1999),Bioprocess Engineering, 21, 453-458; Kaderbhai, M. A., Ugochukwu, C. C.,Lamb, D. C., Kelly, S. (2000) Biochem. Biophys. Res. Comm. 279, 803-807;Yang, J., Moyana, T., Mackenzie, S., Xiz, Q., and Xiang, J. (1998)Applied Environmental Microbiology, 67, 1805-1814). Co-expression ofcellular proteins such as kil, TolAIII, and bacteriocin release protein(BRP) can enhance the release of intracellular protein by providingpores through which the protein can traverse the membrane or byincreasing the permeability of the cell membrane (Zhou, S., Yomano, L.Pl, Saleh, A. Z., Davis, F. C., Aldrich, H. C., Ingram, L. O. (1999)Applied Environmental Microbiology, 65, 2439-2445; Kujau, M. J.,Hoischen, C., Riesenberg, D., Gumpert, J. (1998) Applied MicrobialBiotechnology, 49, 51-58; Van der Wal, F. J., ten Hagen-Jounman, C. M.,Oudega, B., Luirink, J., (1995) Applied Microbial Biotechnology, 44,459-465). Cells deficient in cell walls called L-form cells can also beused to secrete intracellular proteins into the supernatant.

Binding of Biotinylated Protein to the Cell Surface

The interaction between biotin and avidin is extremely tight(K_(d)˜10⁻¹⁵ M) yielding a covalent-like interaction between the twomoieties. A powerful display system for protein engineering is createdby connecting avidin to the surface of a cell and binding a biotinylatedprotein of interest to the avidin. Proteins are immobilized on cellsurfaces through avidin in ways that leave them accessible tomodification or labeling by antibodies that will be used for selectionvia flow cytometry or panning against an immobilized antigen. Avidin canbe covalently conjugated to the cell surface or avidin can be connectedto the cell surface through a spacer.

Connection of Avidin through a Spacer

In yeast and mammalian cells, a biotin may be attached to the cellsurface via a polyethylene glycol linker, facilitated by the presence ofan N-succimidyl ester (NHS) functional group. This NHS functional groupallows the PEG to covalently attach to free amines present on proteinson the cell surface. On the other end of this PEG linker is the biotin.The free biotin binds avidin which, in turn, binds up to three otherbiotinylated proteins due to avidin's tetravalent avidity (FIG. 1).

Covalent Conjugation of Avidin to the cell Surface

Avidin can be covalently conjugated to the cell surface through ahetero-bifunctional connector like C6-SANH (FIG. 2). Yeast cells werelabeled with C6-SANH in carbonate buffered to a pH of 8.4 for 30 minutesat room temperature. Simultaneously, 200 μl of 50 mg/ml avidin wasincubated in 5 mM periodate dissolved in phosphate buffer adjusted to apH of 5.6 for 30 minutes at room temperature. After incubation periodatewas removed using a desalting column, from which the avidin was elutedin phosphate buffer at pH 5.6. The periodate-treated avidin was thenmixed with washed C6-SANH-treated cells for 30 minutes at roomtemperature. After the incubation, the cells were washed three times inPBS/BSA (pH 7.2), then labeled with biotinylated fluorescein to measurehow much avidin was associated with the cell surface. After 20 minuteincubation on ice, the cells were washed and analyzed by flow cytometry(FIG. 3A and FIG. 3B). To show the stability of the avidin-cell surfaceconnection, cells were either labeled immediately with biotinylatedfluorescein (0 min) or incubated in 1 mL PBS/BSA for 30 minutes at roomtemperature before labeling with biotinylated fluorescein (30 min). FIG.3C demonstrates the stability of the avidin-cell linkage over time.

Example 2 Protein Display on the Yeast Surface Using Free Avidin toReplenish Surface Avidin Levels

Yeast strain JKI100 (matα, GAL1 promoter BirA ligase::URA3, GAL1promoter protein disulfide isomerase::LEU2, pep4::HIS, prb1Δ, trp1) wastransformed with CEN plasmid carrying a small, single-domain,FLAG-tagged protein fused to the N-terminus of the BirA biotin acceptorpeptide (BAP). Yeast were grown overnight in 5 mL SD-CAA media at 30° C.The cell culture was spun down and resuspended in 5 mL YPG/bovine serumalbumin +2.5 μg/mL biotin supplement and shaken for six hours at 30° C.Approximately 1×10⁷ cells were removed and mixed at a ratio of 1:300with cells not expressing the protein-BAP fusion (1 expressing cell forevery 300 non-expressing cells), and washed three times in 1 mLcarbonate buffer (pH=8.4). The mixture of cells was resuspended in 40 μl0.1 mg/ul NHS-PEG-biotin in carbonate buffer and incubated for 30minutes at room temperature.

Cells were then washed three times in 1 mL PBS/BSA and incubated in 50ul of 20 mg/mL avidin for ten minutes twice. Cells were inoculated into3 mL biotin-less display media (made by mixing 30 μl of 100 mg/mL avidinwith 3 mL filter sterilized biotin-less YPG/BSA/PEG (30 wt %)) andincubated in 2 wells of a six well plate overnight at 30° C. Cells werewashed off the bottom of the well with 1 mL PBS/BSA and spun down andwashed three times with 1 mL cold PBS/BSA. Cells were then incubated in50 μl of 1:50 dilution chicken anti FLAG antibody for 20 minutes on ice.After washing in 500 μl PBS/BSA, cells were incubated in 50u1 1:100dilution goat anti-chicken Alexa633 and 50nM biotin-fluorescein for 20minutes on ice. After labeling, cells were washed once in 500 μl PBS/BSAand run on FACS.

The non-expressing cells were used as an internal negative control todemonstrate that protein capture (exhibited by a FLAG-positivepopulation on the flow cytometer) was limited to cells expressing theplasmid for the protein-BAP fusion. To demonstrate the specific linkagebetween FLAG expression and the presence of plasmid, the avidinpositive/FLAG positive population was sorted directly onto YPD plates (anon-selective, rich media). Two days later the colonies were replicateplated onto media only selective for cells that contain the FLAG/BAPfusion plasmid. It was demonstrated that plasmid positive cells wereenriched to 10% of the total population in the sorted cells for anoverall enrichment of 30-fold. This experiment shows that the displaymethod described here does maintain the phenotype/genotype linkagenecessary for protein engineering applications.

Example 3 Surface Display and Selection of IgG

The usefulness of the display system in isolating proteins that can binda specific antigen was tested in a “mock selection”. Yeast strain JKI100(mata, GAL1 promoter BirA ligase::URA3, GAL1 promoter protein disulfideisomerase::LEU2, pep4::HIS, prb1Δ, trp1) was transformed with a CENplasmid carrying both chains of one of two IgGs (some cells transformedwith IgG-A and some transformed with IgG-B). In these constructs theheavy chain is fused to the N-terminus of the biotin acceptor peptide(BAP) and the light chain is fused to the C-terminus of a FLAG tag.Yeast were grown overnight in 5 mL SD-CAA media at 30° C. The culturewas spun down and resuspended in 5 mL YPG/bovine serum albumin +2.5μg/mL biotin supplement and grown under shaking for seven hours at 20°C. The cells were removed and mixed in a ratio of 1:10,000 IgG-A toIgG-B (one IgG-A expressing cell for every 10,000 IgG-expressing cell).One OD₆₀₀ mL of the mixture (˜1×10⁷ cells) was removed and washed threetimes in 1 mL carbonate buffer (pH=8.4). Cells were resuspended in 40μmg/μl NHS-PEG-biotin in carbonate buffer and incubated for 30 minutesat room temperature. After three washes in 1 mL PBS/BSA cells wereincubated in 50 μl of 20 mg/mL avidin for ten minutes, twice. Cells wereinoculated into 3 mL biotin-less display media YPG/BSA/PEG (30 μl of100mg/mL avidin with 3 mL biotin-less filter-sterilized YPG/BSA/PEG (10wt % PEG)) and incubated in 2 wells of a six well plate overnight at 20°C.

Cells were washed off the bottom of the well with 1 mL PBS/BSA and spundown then washed three times with 1 mL cold PBS/BSA. Cells wereincubated in a 1:20 dilution of 1 uM His6 tagged IgG-A antigen. Cellswere then labeled by incubation in 50 ul 1:50 dilution chicken anti FLAGantibody for 20 minutes on ice and 1:50 dilution of mouse anti-His6antibody. Cells were washed once in 500 μl PBS/BSA and then incubated in50 μl 1:100 dilution goat anti-chicken Alexa488 and 1:30 dilution ofgoat anti-mouse PE for 20 minutes on ice. After a wash in 500 μlPBS/BSA, cells binding IgG-A antigen were sorted by FACS and collectedin 5 mL SD-CAA. The cells were expanded, induced, put through theselection assay, and then sorted an additional time for a total of tworounds of selection. Cells from the final sort were expanded, and theirDNA prepped for sequencing. Sequencing analysis showed that the IgG-Aclone had been enriched 9,800-fold over the IgG-B expressing clone. Acontrol consisting of an equimolar mixture of the clones in which allIgG expressing cells were taken in the two rounds of selection was alsoperformed to normalize for IgG-A selection that might have occurredindependent of the surface display and sorting. This control showed thatno such selection occurred, and the enrichment was due to the IgG-Aantigen labeling only.

Example 4 Surface Display of ScFv

Yeast strain JKI100 (matα, GAL1 promoter BirA ligase::URA3, GAL1promoter protein disulfide isomerase::LEU2, pep4::HIS, prb1Δ, trp1) wastransformed with a CEN plasmid carrying a single chain antibodyrecognizing either antigen A or antigen B. The fusion protein wasengineered to have a biotin acceptor peptide fused at the C-terminus ofthe variable domain light chain which is fused to the C-terminus of thevariable domain heavy chain fused to the C-terminus of a FLAG tag.

After transformation, the yeast transformants were mixed in a ratio of 1cell of antigen-A binding scFv to 10,000 antigen-B binding cells. Themixture followed the same treatments, rounds of selection, and controlsas those in Example 3. Antigen A-binding yeast cells were enriched110-fold over two rounds of FACS.

Example 5 Surface Display of IgG with Nocodozole Treatment

Nocodozole treatment is performed to increase the percentage of avidinlabeled cells in a population by inhibiting cell cycle. Yeast strainJKI100 (mata, GAL1 promoter BirA ligase:: URA3, GAL1 promoter proteindisulfide isomerase::LEU2, pep4::HIS, prb1Δ, trp1) was transformed witha CEN plasmid carrying both chains of an IgG. The heavy chain is fusedto the N-terminus of the biotin acceptor peptide (BAP) and the lightchain is fused to the C-terminus of a FLAG tag. Yeast were grownovernight in 5 mL SD-CAA media at 30° C. The culture was spun down andresuspended in 5 mL YPG/bovine serum albumin and 2.5 μg/mL biotinsupplement and grown under shaking for seven hours at 20° C. One OD₆₀₀mL of cells (˜1×10⁷ cells) was removed and washed three times in 1 mLcarbonate buffer (pH=8.4). Cells were resuspended in 40 μl 0.1 mg/μlNHS-PEG-biotin in carbonate buffer and incubated for 15 minutes at roomtemperature. After three washes in 1 mL PBS/BSA, cells were incubated in50 μl of 20 mg/mL avidin for ten minutes. 0.3 mL of cells wereinoculated into 1.2 mL biotin-less display media YPG/BSA/PEG (30 μl 100mg/mL avidin with 3 mL biotin-less filter-sterilized YPG/BSA/PEG (10 wt% PEG)) with 0 μg/mL nocodozole or 20 μg/mL nocodozole and incubated in2 wells of a six well plate overnight at 20° C. Cells were washed offthe bottom of the well with 1 mL PBS/BSA and spun down then washed threetimes with 1 mL cold PBS/BSA. Cells were incubated in a 1:20 dilution of1 uM His6 tagged antigen. Cells were then labeled by incubation in 50 μl1:50 dilution chicken anti FLAG antibody for 20 minutes on ice and 1:50dilution of mouse anti His6 antibody. Cells were washed once in 500 μlPBS/BSA and then incubated in 50 μl 1:100 dilution goat anti-chickenAlexa488 and 1:30 dilution of goat anti-mouse PE for 20 minutes on ice.In addition to the FLAG and antigen labeling described above, cells werelabeled with 50 μl 50 nM biotin-fluorescein to test what fraction ofyeast cells possesses avidin on their surface.

30% of the cells not treated with nocodazole do not possess surfacelabeled avidin. These cells are probably daughter cells that did notinherit the avidin from their mothers. The cells treated with 20 ug/mLnocodazole possessed only 5% unlabeled avidin cells (FIGS. 12A-12D).Labeling with the antigen and anti-FLAG antibody showed that theexpression and binding ability of the displayed protein was minimallyperturbed by the treatment. In addition, similar experiments werecarried using treatments of hydroxyurea at 200 mM and 50 mMconcentrations, EGTA at 5 mM concentration, and farnesol at 25 uM. Thesetreatments also retarded cell division by varying amounts withoutsignificantly impacting expression or function of the displayed protein.

Example 6 Surface Expression of Biotinylated Cell Wall Protein

Yeast strain JKI100 (mata, GAL1 promoter BirA ligase:: URA3, GAL1promoter protein disulfide isomerase::LEU2, pep4::HIS, prb1Δ, trp1) wastransformed with a CEN plasmid carrying a mutant of the cell wallprotein SAG1 containing a biotin acceptor peptide in the extracellulardomain (FIG. 4B). Yeast were grown overnight in 5 mL SD-CAA media at 30°C. The culture was spun down and resuspended in 5 mL YPG/bovine serumalbumin and 2.5 μg/mL biotin supplement and grown under shaking forseven hours at 20° C. After an overnight incubation at 30° C., the cellswere washed three times with 1 mL cold PBS/BSA. Cells were incubated in20 mg/mL avidin for ten minutes, twice, then washed once in 1 mLPBS/BSA. Cells were then labeled with 50 μl 50 nM biotin-fluorescein andanalyzed by flow cytometry to highlight cells displaying thebiotinylated SAG1 protein (P4, FIG. 4D). Because CEN plasmids areunstable in yeast, a significant portion of the cells do not display thebiotinylated SAG1 protein. These cells are the negative peak in the FIG.4D. This instability also serves as an internal negative control todemonstrate that avidin and biotin-fluorescein labeling is exclusive tocells expressing the biotin-SAG1 protein.

Example 7 Selection of Thermostable Protein Mutants

Yeast strain JKI100 (mata, GAL1 promoter BirA ligase:: URA3, GAL1promoter protein disulfide isomerase::LEU2, pep4::HIS, prb1Δ, trp1) wastransformed with a CEN plasmid carrying one of two small, single domainprotein mutants. One mutant has a melting temperature of 42° C. asdetermined by differential scanning calorimetry. The other mutant has amelting temperature of 82° C. determined by DSC. Both of these mutantproteins were expressed as C-terminal fusions to the BAP biotin acceptorpeptide and N-terminal fusions to the FLAG tag.

After transformation, the yeast transformants were mixed in a ratio 1cell of the more thermostable mutant to 100 cells of the lessthermostable mutant. The mixture followed the same treatments, rounds ofselection, and controls as those in Example 3 except only FLAG tag waslabeled and used as a criterion for selection. After two rounds ofselection, isolated clones were sequenced, and it was determined thatthe more thermostable clone was enriched 45-fold over the lessthermostable clone (FIGS. 10A and 10B).

Example 8 DNA Attachment to Cell Surface for Directed Evolution of DNAModifying Enzymes

Directed Evolution has been a successful strategy for engineeringproteins with enhanced binding or activity characteristics. Mostdirected evolution of DNA modifying proteins such as polymerases hasbeen done in bacteriophage or in emulsions of cell lysate (Tawfik D,Griffiths A. “Man-made Cell-like Compartments for Molecular Evolution.”Nature Biotechnology 1998, 16: 652-656; Ghadessy F., Ong, J., Holliger,P., “Directed Evolution of Polymerase Function by compartmentalizedSelf-replication.” PNAS USA 2001, 98: 4552-4557; Ong, J. L., Loakes, D.,Jaroslawski, S., Too, K., Holliger, P., “Directed Evolution of DNAPolymerase, RNA Polymerase, and Reverse Transcriptase Activity in aSingle Polypeptide.” Jour. Mol. Biol. (2006), 361: 537-550; Jestin, J.L., Kristensen, P., Winter, G. “A Method for the Selection of CatalyticActivity Using Phage display and Proximity Coupling.” Angew Chem. Int EdEngl 1999, 38: 1124-2237; Brunet, E., Chauvin, C., Choumet, V., Jestin,J. L. “A Novel Strategy for the Functional Cloning of Enzymes UsingFilamentous Phage Display: the Case of Nucleotidyl Transferases.”Nucleic Acids Res 2002, 30:e40; Xia, G., Chen, L., Sera, T., Fa, M.,Schultz, P., Romesberg, F. “Directed Evolution of Novel PolymeraseActivities: Mutation of a DNA Polymerase into an efficient RNAPolymerase.” PNAS USA 2002, 99: 6597-6602; Fa, M., Radeghieri, A.,Henry, A., Romesberg, F. “Expanding the Substrate Repertoire of a DNAPolymerase by Directed Evolution.” J. American Chem. Society 2004, 126:1748-1754). In the former case, selection relies on the ability of thepolymerase to incorporate a biotinylated substrate. In the latter case,the selection relies on the ability of the polymerase to completelytranscribe its own gene while incorporating novel base pairs orcatalytic activity. Consequently, these approaches are limited to caseswhere biotinylated dNTP incorporation or polymerase processivity isessential, or at least tolerated. A new selection system is presented,which overcomes earlier limitations and allows for the selection ofpolymerases that use many different dNTPs, or to select for polymeraseswith lower processivity. The technique outlined in FIGS. 13-16 links aDNA oligo to cells via a high affinity biotin/avidin interaction. Onceattached to the cell, the DNA can interact with enzymes that aredisplayed on the cell surface as well or that are secreted into thesupernatant.

Nucleic acid Attachment to the Cell Surface (FIG. 13)

To use nucleic acid in display-based screening, nucleic acid isphysically attached to the cell. The biotin/avidin interaction, beingextremely high affinity (K_(d)˜10⁻¹⁵ M), is well suited to attach DNA tothe cell surface. The biotin/avidin interaction has been used previouslyto immobilize proteins on the cell surface (Manz, R., Assenmacher, M.,Pfluger, E., Miltenyi, S., Radbruch, A. “Analysis and Sorting of LiveCells According to Secreted Molecules, Relocated to Cell-SurfaceAffinity Matrix.” (1995) PNAS, 92(6): 1921-1925; Rakestraw A., Baskaran,A., Wittrup, K. D., “A Flow Cytometric Assay for Screening ImprovedHeterologous Protein Secretion in Yeast.” (2006) Biotechnology Progress,22(4): 1200-1208). Cells are labeled with biotin using a polyethyleneglycol (PEG) linker. In one form this linker possesses biotin on one endand a free amine reactive N-succimidyl ester (NHS) group on the other.In this manner biotin can be directly conjugated to proteins on the cellsurface in an alkaline buffer. Biotin can also be attached tocarbohydrate on the cell wall, thereby preserving the integrity of cellsurface proteins. For this alternative the cells are treated withperiodate (a compound that opens the carbohydrate ring and oxidizesadjacent hydroxyls into aldehydes) or an enzyme, such as galactoseoxidase, that similarly but non-chemically opens carbohydrate ringstructures. After treatment, the cells are labeled withbiotin-polyethylene glycol-hydrazide which covalently links the biotinto the surface via the exposed aldehydes on the oxidized sugars. Afterlabeling the cells with NHS-PEG-biotin or hydrazide-PEG-biotin, thelabeled cells are exposed to avidin resulting in the binding of avidinto biotin. Because avidin is tetravalent, it can accept up to threeadditional biotin molecules. Nucleic acid strands can therefore be boundto the avidin by biotin conjugated to the 3′ or 5′ ends of a nucleicacid oligomer. For example, if a single stranded DNA oligomer isattached to avidin, the complementary strand or primer can be annealed,or modifications using single strand DNA as a substrate can beperformed. Annealing of the complementary strand generates doublestranded DNA, which can be subjected to enzymatic processes such asrestriction cleavage or primer extension (FIG. 15 and FIGS. 16A and16B). Immobilized DNA allows for the evaluation of a variety of enzymes.For instance polymerase activity can be detected by the incorporation offluorescent oligonucleotides. The use of fluorescent complementaryoligos or dNTPs, allows for the screening of enzyme activity of bothsurface displayed proteins and proteins in the supernatant. Cells can beisolated by flow cytometry allowing for the selection of enzymes withoptimized properties. A variety of different moieties can be attached tothe cell surface via the avidin linkage, including biotinylated RNA,peptides, and full length protein. This methodology thus provides ameans to evaluate many different types of protein and enzymaticinteractions on the surface of cells.

Biotinylated Oligo Attachment

Yeast were labeled with NHS-PEG-biotin and avidin in preparation foroligonucleotide attachment. A 5′ biotinylated single-stranded oligo wasadded to be immobilized on the yeast surface via the avidin sandwich.The attached oligonucleotide was detected by annealing a FAM(6-carboxyfluorescein) fluorescent labeled complementary strand to thecell surface. Flow cytometry data for a complementary andnon-complementary FAM labeled oligonucleotide are shown in FIGS. 14A and14B.

On Cell Restriction Digest

The annealed double stranded oligo contains an I-SceI endonuclease site.The cells were incubated with I-SceI and analyzed by flow cytometry. Asthe DNA was cleaved, the fluorophores was released from the surfacecausing a decrease in FAM signal over time (FIG. 15). Treatment withNheI was used as a negative control.

On Cell Primer Extension

A small complementary primer was annealed to an oligo, which wasimmobilized on the surface of a yeast cell. The yeast were subsequentlyincubated in PCR buffer containing dNTPs including 25 μM dUTP-Cy5fluorescent dye. When Klenow fragment polymerase was added to themixture, the primer was extended allowing the surface construct toincorporate the fluorescent dye as indicated by flow cytometry (FIG.16B).

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by examples provided, since theexamples are intended as a single illustration of one aspect of theinvention and other functionally equivalent embodiments are within thescope of the invention. Various modifications of the invention inaddition to those shown and described herein will become apparent tothose skilled in the art from the foregoing description and fall withinthe scope of the appended claims. The advantages and objects of theinvention are not necessarily encompassed by each embodiment of theinvention.

The contents of all references, patents and published patentapplications cited throughout this application are incorporated hereinby reference in their entirety.

1. A protein screening method comprising: a. expressing an engineeredprotein comprising a modification motif in a host cell having a cellsurface comprising a first binding partner, wherein a second bindingpartner is coupled to the expressed engineered protein, and wherein theexpressed engineered protein is secreted and displayed on the cellsurface via binding of the second binding partner to the first bindingpartner; and, b. evaluating a property of the engineered proteindisplayed on the cell surface.
 2. The method of claim 1, wherein theevaluating step comprises assaying a level of activity, determiningwhether the engineered protein has a predetermined function, comparingthe property of the engineered protein to the property of a referenceprotein, determining the amount of the engineered protein displayed onthe cell surface, or any combination thereof.
 3. The method of claim 1,wherein the engineered protein is an antibody, a single chain antibody,a scaffold protein, or a fragment thereof, and the function of theprotein being evaluated is the binding affinity of the protein to thetarget molecule, wherein the target molecule comprises an antigen and/orepitope.
 4. The method of claim 1, wherein host cells are selected onthe basis of a first predetermined property of the displayed engineeredprotein.
 5. The method of claim 4, further comprising selecting the hostcells on the basis of a second predetermined property of the displayedengineered protein.
 6. The method of claim 4, further comprisingreleasing the engineered protein from selected host cells displaying atleast the predetermined level of the engineered protein.
 7. The methodof claim 2, wherein assaying a level of activity comprises assaying ifthe engineered protein can process a substrate.
 8. The method of claim7, wherein the substrate is coupled to the surface.
 9. The method ofclaim 7, wherein the substrate is a polypeptide, nucleic acid, lipid,polysaccharide, synthetic polymer or synthetic compound.
 10. The methodof claim 7, wherein processing a substrate comprises binding to thesubstrate, dissociating the substrate, nicking the substrate, cuttingthe substrate, activating the substrate, deactivating the substrate,charging the substrate, decharging the substrate, changing substrateconformation, copying the substrate, replicating the substrate,conjugating molecules to the substrate, conjugating peptides to thesubstrate or modifying the substrate.
 11. The method of claim 2, furthercomprising isolating an engineered protein having a predeterminedfunction.
 12. The method of claim 2, further comprising isolating anengineered protein having a predetermined level of activity.